Di-isopropyl-phosphinoyl-alkanes (dapa) compounds as topical agents for the treatment of sensory discomfort

ABSTRACT

The present invention pertains generally to the field of therapeutic compounds. More specifically the present invention pertains to certain di-isopropyl-phosphinoyl-alkanes as described herein (DIPA-1-5, DIPA-1-6, DIPA-1-7, DIPA-1-8, and DIPA-1-9, collectively referred to herein as “DIPA compounds”) that are useful, for example, in the treatment of disorders (e.g., diseases) including: sensory discomfort (e.g., caused by irritation, itch, or pain); a skin dysesthesia; dermatitis; psoriasis; ocular discomfort; heat discomfort; heat stress; flushing and/or night sweats (vasomotor symptoms) in post-menopausal women; post-operative hypothermia; post-anaesthetic shivering; fatigue; tiredness; depression; cognitive dysfunction; and to enhance cognitive function. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, for example, in therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/350,559 filed on Nov. 30, 2018, which is a continuation-in-partof U.S. application Ser. No. 14/544,355, filed on Dec. 29, 2014, nowU.S. Pat. No. 10,195,217 B2, which is a continuation-in-part ofPCT/GB2013/052,750, filed on Oct. 22, 2013.

TECHNICAL FIELD OF THE INVENTION

The present invention pertains generally to the field of therapeuticcompounds. More specifically the present invention pertains to certaindi-isopropyl-phosphinoyl-alkanes as described herein (DIPA-1-6,DIPA-1-7, DIPA-1-8, and DIPA-1-9, collectively referred to herein as“DIPA compounds”) that are useful, for example, in the treatment ofdisorders (e.g., diseases) including: sensory discomfort (e.g., causedby irritation, itch, or pain); a skin dysesthesia; dermatitis;psoriasis; ocular discomfort; heat discomfort; heat stress; flushingand/or night sweats (vasomotor symptoms) in post-menopausal women;post-operative hypothermia; post-anaesthetic shivering; fatigue;tiredness; depression; cognitive dysfunction; and to enhance cognitivefunction. The present invention also pertains to pharmaceuticalcompositions comprising such compounds, and the use of such compoundsand compositions, for example, in therapy.

BACKGROUND OF THE INVENTION

A number of publications are cited herein in order to more fullydescribe and disclose the invention and the state of the art to whichthe invention pertains. Each of these publications is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art or relevant to the presentlyclaimed invention, or that any publication specifically or implicitlyreferenced is prior art.

Chemical Cooling Agents

Air blown onto the face from a fan or an air-conditioner can reducetiredness and increase alertness. A wet towel applied to the foreheadcan relieve discomfort from a fever or a headache. These methods achievetheir effects by physically lowering tissue temperatures and activatingsignals to the brain with the message that the external environment iscool.

A chemical which produces cooling/cold sensations on facial skin withoutchanging tissue temperatures might achieve the same objectives. The term“chemical cooling agent” can be ambiguous because, for example,chemicals such as ethanol or ethyl chloride applied to the skin causeevaporative cooling and a reduction of tissue temperatures.

The inventor has identified compounds that, when applied to the skin,potently simulate effects of heat abstraction without a decrease intissue temperature (see, e.g., Wei 2012). The effects are observed atapplied doses of less than 5 mg, and the level of robust and intensecooling achieved on the skin is unusual and has not been previouslyrecognized.

It has been known for some time that an environmental temperature below21.1° C. (70° F.) is optimal for work performance, and that the besttemperature is in the range of 18.3 to 20° C. (65 to 68° F.) (see, e.g.,Dawson et al., 2009). Experimentally, an improvement in performance canbe demonstrated at 20° C. versus a 23° C. environment (see, e.g., Thamand Willem, 2010). Thus, an optimal cool environment reduces fatigue andimproves work output. By localizing the dynamic cooling effect to thefacial skin surrounding the eyes and on the margins of the eyelids, theinventor has found that this alerting and enhancement effect can befocused and magnified.

The skin of the face and the orbit are especially sensitive tothermosensory information and a drop in ambient temperature below 15 to18° C. activates brain structures and pathways for arousal/vigilance.The inventor proposed that the application of a sensory agent thatevokes a sensation of “dynamic cool” will arouse an organism andcounteract tiredness. This change in mind-set is the basis of thechemically-induced anti-fatigue effect. The strategy is that of atopical skin sensory agent, and not that of an ophthalmic product.

Feeling tired, weary, and fatigued is a common experience and isconsidered an inconvenience that may be resolved by taking a nap,drinking a cup of coffee, or stopping whatever activity that brought iton. In many disorders, however, fatigue is a non-specific symptom withadverse consequences.

Fatigue, and its operational deficits, are recognized in this definitionby the Federal Aviation Administration: “Fatigue is a conditioncharacterized by increased discomfort with lessened capacity for work,reduced efficiency of accomplishment, loss of power or capacity forwork, reduced efficiency of accomplishment, loss of power or capacity torespond to stimulation, and is usually accompanied by a feeling ofweariness and tiredness” (see, e.g., Salazar, 2013).

Conditions that cause fatigue include: anxiety, boredom, depression,disruption of circadian rhythm or sleep, heavy physical exertion,excessive mental activity, treatment for cancer, chronic illness, andheat stress (see, e.g., Salazar, 2013; Stasi et al., 2003). Thedefinition used by the National Cancer Institute for fatigue is acondition marked by extreme tiredness and inability to function due lackof energy. Fatigue may be acute or chronic (greater than 1 monthduration), and, depending upon the accompanying symptoms, severity, andduration, it may be further classified as mild, moderate, or severe.Fatigue is a subjective sensation and its primary symptom is a complaintof tiredness. See, e.g., National Cancer Institute, 2013.

Drugs such as caffeine, amphetamines, methylphenidate, nicotine,donepezil, and modafinil have been used to treat fatigue. Thesecompounds act invasively on brain chemistry.

That is, the drugs require access of the active agent to thebloodstream, and from there to central nervous system, to act uponenzymes or receptors. Drugs such as amphetamines and nicotine haveaddiction liability. Even caffeine can over-stimulate the nervous systemand causes palpitations, irritability, tolerance, and dependence. Thereis a need for alternative methods for the treatment of tiredness andfatigue.

A further effect that has been observed for the compound describe hereinis a potent suppression of sensory discomfort on irritated, itching, orpainful keratinized skin. This activity on the skin has applications inthe treatment of skin disorders, especially for irritation, itch, andpain.

Known Phosphine Oxides

Rowsell et al., 1978, describes a range of phosphine oxides which have aphysiological cooling effect on skin and on the mucous membranes of thebody, particularly the nose, mouth, throat and gastrointestinal tract.See, e.g., the table in columns 3 and 4 therein. Ten (10) of thecompounds shown therein (see the following table) have one isopropylgroup (shown as iso-C3H7). None of the compounds is DIPA-1-6, DIPA-1-7,DIPA-1-8, or DIPA-1-9. Indeed, none of the compounds has two isopropylgroups.

Compounds in Rowsell et al., 1978 P( = O)R¹R²R³ # R¹ R² R³  2 n-C₇H₁₅iso-C₃H₇ sec-C₄H₉  3 n-C₈H₁₇ iso-C₃H₇ sec-C₄H₉  7 n-C₆H₁₃ iso-C₃H₇sec-C₄H₉  8 n-C₆H₁₃ iso-C₃H₇ cyclo-C₅H₉ 11 n-C₇H₁₅ iso-C₃H₇ cyclo-C₅H₉12 n-C₆H₁₃ iso-C₃H₇ iso-C₅H₁₁ 15 n-C₇H₁₅ iso-C₃H₇ iso-C₅H₁₁ 26 n-C₆H₁₃iso-C₃H₇ n-C₆H₁₃ 30 n-C₈H₁₇ iso-C₃H₇ cyclo-C₅H₉ 47 iso-C₃H₇ n-C₄H₉(n-C₄H₉)(C₂H₅)CHCH₂

Wei, 2005, describes the use of certain phosphine oxides and thetreatment of eye discomfort by the administration of eye dropscontaining those compounds. See, e.g., Table 1 on page 4 therein. Five(5) of the compounds shown therein (see the following table) have oneisopropyl group (shown as iso-C₃H₇). None of the compounds is DIPA-1-6,DIPA-1-7, DIPA-1-8, or DIPA-1-9. Indeed, none of the compounds has twoisopropyl groups.

Compounds in Wei, 2005 P( = O)R¹R²R³ # R¹ R² R³ 14 n-C₆H₁₄ iso-C₅H₁₁iso-C₃H₇ 15 n-C₇H₁₅ iso-C₅H₁₁ iso-C₃H₇ 17 n-C₆H₁₄ iso-C₃H₇ sec-C₄H₉ 18n-C₇H₁₅ iso-C₃H₇ sec-C₄H₉ 19 n-C₈H₁₇ iso-C₃H₇ sec-C₄H₉

To date, neither the preparation of, nor the evaluation of DIPA-1-6,DIPA-1-7, DIPA-1-8, or DIPA-1-9 has been reported.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a human head, showing facial sites fortesting: (a) infraorbital, (b) buccal cheek, (c) zygomatic, (d)parotid-masseteric cheek, (e) frontal, and (f) periorbital. Taken fromPilsl et al., 2012.

FIG. 2 is a graph of response (Relative Fluorescence Units; % ofmaximum) as a function of the logarithm of the concentration of the testcompound (denoted agonist), expressed in μM, for each of 1-5 (circles),DIPA-1-6 (squares), DIPA-1-7 (inverted triangle), DIPA-1-8 (diamonds),or DIPA-1-9 (up-right triangle).

FIG. 3 shows chart traces that illustrate, in the first trace (“WildType”), the inhibition of capsaicin-induced depolarization of theisolated mouse vagus by DIPA-1-7, superfused at a concentration of 1mg/mL, and, in the second trace (“TRPM8 KO”), the significant absence ofinhibition in the isolated TRPM8 KO (knockout) mouse vagus by DIPA-1-7,superfused at a concentration of 1 mg/mL.

SUMMARY OF THE INVENTION

One aspect of the invention pertains to certaindi-isopropyl-phosphinoyl-alkanes described herein (collectively referredto herein as “DIPA compounds”).

Another aspect of the invention pertains to a composition (e.g., apharmaceutical composition) comprising a DIPA compound, as describedherein, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention pertains to a method of preparing acomposition (e.g., a pharmaceutical composition) comprising the step ofmixing a DIPA compound, as described herein, and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the present invention pertains to a DIPA compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy, for example, for use a method of treatment of adisorder (e.g., a disease) as described herein.

Another aspect of the present invention pertains to use of a DIPAcompound, as described herein, in the manufacture of a medicament fortreatment, for example, treatment of a disorder (e.g., a disease) asdescribed herein.

Another aspect of the present invention pertains to a method oftreatment, for example, of a disorder (e.g., a disease) as describedherein, comprising administering to a patient in need of treatment atherapeutically effective amount of a DIPA compound, as describedherein, preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to a kit comprising (a)a DIPA compound, as described herein, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the compound.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to certain compounds (the DIPA compoundsdescribed herein) which, when delivered onto the facial skin, especiallyon the periorbital and malar surfaces, selectively and potently evokesensations of “dynamic cool” for at least several hours. The DIPAcompounds may be used to counteract fatigue and enhance cognitivefunction. The DIPA compounds are administered topically, and so effectsare achieved without direct invasion of brain chemistry. The dynamiccool can be repeated without significant diminution of the effects andcan be sustained for the whole day. The sensations on the facial skin donot interfere with the individual's ability to fall asleep. The DIPAcompounds have applications in the treatment of skin discomfort,especially skin irritation, itch, and pain. The DIPA compounds may alsobe especially useful to counter fatigue from heat stress, chronicillness, or to enhance work performance. The DIPA compounds may also beused to counteract the flushing and “night sweats” (vasomotor symptoms)that occur in post-menopausal women.

DIPA Compounds

The compounds of the present invention are examples of phosphine oxides(which have the following general formula), and more particularly, areexamples of di-alkyl-phosphinoyl-alkanes (wherein each of R¹, R², and R³is an alkyl group).

More specifically, the compounds of the present invention are thefollowing compounds (collectively referred to herein as “DIPAcompounds”):

Formula/ Code Chemical Name Weight Chemical Structure DIPA- 1-61-di(isopropyl)- phosphinoyl-hexane C₁₂H₂₇OP 218.32

DIPA- 1-7 1-di(isopropyl)- phosphinoyl-heptane C₁₃H₂₉OP 232.34

DIPA- 1-8 1-di(isopropyl) phosphinoyl- octane C₁₄H₃₁OP 246.37

DIPA- 1-9 1-di(isopropyl) phosphinoyl- nonane C₁₅H₃₃OP 260.40

DIPA-1-7 is a colorless liquid with a density of ^(˜)0.85 g/cm3. It isreadily soluble in water or saline at up to 20 mg/mL. When it is appliedto the facial skin as an aqueous solution at 1-10 mg/mL there is littleirritation. Contacting the periorbital, infraorbital, or malar skin witha solution at a concentration of 1-10 mg/mL produces a sensation of“dynamic cool” that is felt within one minute after application.Following a single application at a concentration of 1-10 mg/mL, thissensation counteracts fatigue for five or more hours.

The potent sensory effects of DIPA-1-7 and DIPA-1-8 are surprisinglyspecific and not seen with structurally similar analogs. DIPA-1-8 islonger-acting than DIPA-1-7, but it has a lower dynamic coolingintensity. Both DIPA-1-7 and DIPA-1-8 (and in particular DIPA-1-7) areespecially useful for treatment of skin dysesthesias (e.g., skinirritation, itchy skin, or painful skin), ocular discomfort, heatdiscomfort, and heat stress.

DIPA-1-9 causes the least amount of irritation, and so is especiallyuseful for the treatment of ocular discomfort, possibly evenadministered as eye drops. DIPA-1-6 does not act for as long asDIPA-1-7, but is absorbed more easily across the skin, and is thereforeespecially useful for systemic applications, e.g., in the treatment offlushing and/or night sweats (vasomotor symptoms) in post-menopausalwomen.

Chemical Synthesis

The DIPA compounds were prepared by the following general method: 100 mL(23.7 g, ^(˜)200 mmol) of isopropylmagnesium chloride (orsec-butylmagnesium chloride in the case of the di-sec-butyl derivatives)were obtained from Acros, as a 25% solution in tetrahydrofuran (THF) andplaced under nitrogen in a 500 mL flask (with a stir bar).Diethylphosphite solution in THF (from Aldrich, D99234; 8.25 g, 60.6mmol in 50 mL) was added drop-wise. After approximately 30 minutes, thereaction mixture warmed up to boiling. The reaction mixture was stirredfor an extra 30 minutes, followed by a drop-wise addition of theappropriate n-alkyl iodide solution in THF (from TCI; 60 mmol in 20 mL).The reactive mixture was then stirred overnight at room temperature. Thereaction mixture was diluted with water, transferred to a reparatoryfunnel, acidified with acetic acid (^(˜)10 mL), and extracted twice withether. The ether layer was washed with water and evaporated (RotaVapBuchi, bath temperature 40° C.). The light brown oil was distilled underhigh vacuum. The final products, verified by mass as determined by massspectrometry, were transparent liquids that were colourless or slightlypale yellow.

The following compounds were prepared by this method:

Code Chemical Name Chemical Structure 1-5 1-di(isopropyl)-phosphinoyl-pentane

DIPA-1-6 1-di(isopropyl)- phosphinoyl-hexane

DIPA-1-7 1-di(isopropyl)- phosphinoyl-heptane

DIPA-1-8 1-di(isopropyl) phosphinoyl- octane

DIPA-1-9 1-di(isopropyl) phosphinoyl- nonane

2-4 1-di(sec-butyl) phosphinoyl- butane

2-5 1-di(sec-butyl) phosphinoyl- pentane

2-6 1-di(sec-butyl) phosphinoyl- hexane

2-7 1-di(sec-butyl) phosphinoyl- heptane

2-8 1-di(sec-butyl) phosphinoyl- octane

3-1 1-di(iso-butyl) phosphinoyl- pentane

3-2 1-di(sec-butyl) phosphinoyl- 3-methyl-butane

Compositions

One aspect of the present invention pertains to a composition (e.g., apharmaceutical composition) comprising a DIPA compound, as describedherein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

Another aspect of the present invention pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingmixing a DIPA compound, as described herein, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

In one embodiment, the composition comprises the DIPA compound at aconcentration of 0.005-2.0% wt/vol.

In one embodiment, the composition is a liquid or semi-liquidcomposition (lotion, cream, or ointment), and comprises the DIPAcompound at a concentration of 0.5-20 mg/mL.

In one embodiment, the composition is a liquid composition, andcomprises the DIPA compound at a concentration of 1-5 mg/mL.

In one embodiment, the composition is a liquid composition, andcomprises the DIPA compound at a concentration of 5-10 mg/mL.

In one embodiment, the composition is a liquid composition, andcomprises the DIPA compound at a concentration of 10-20 mg/mL.

The composition may be provided with suitable packaging and/or in asuitable container.

For example, the composition may be provided as a swab, wipe, pad, ortowellette (e.g., suitably sealed in a wrap) carrying a DIPA compound ora composition comprising a DIPA compound.

Similarly, the composition may be provided as a patch, e.g., acontrolled-release patch, e.g., suitable for application to the skin,e.g., the skin above the supraclavicular fossa or the steronomastoidmuscle.

Similarly, the composition may be provided as an aerosolized spraydelivered from a pressurized container.

Similarly, the composition may be provided in a manually-activatedsprayer (e.g., with a suitable small orifice) linked to a reservoircontaining a DIPA compound or a composition comprising a DIPA compound,for example, capable of delivering an unit volume (e.g., of 0.05 to 0.15mL), for example, to the skin surface.

Use in Methods of Therapy

Another aspect of the present invention pertains to a DIPA compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy, for example, for use a method of treatment of adisorder (e.g., a disease) as described herein.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of a DIPAcompound, as described herein, in the manufacture of a medicament, forexample, for use in a method of treatment, for example, for use a methodof treatment of a disorder (e.g., a disease) as described herein.

In one embodiment, the medicament comprises the DIPA compound.

Methods of Treatment

Another aspect of the present invention pertains to a method oftreatment, for example, a method of treatment of a disorder (e.g., adisease) as described herein, comprising administering to a subject inneed of treatment a therapeutically-effective amount of a DIPA compound,as described herein, preferably in the form of a pharmaceuticalcomposition.

Disorders Treated

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of: sensory discomfort (e.g., caused by irritation, itch, orpain); a skin dysesthesia; dermatitis; psoriasis; ocular discomfort;heat discomfort; heat stress; flushing and/or night sweats (vasomotorsymptoms) in post-menopausal women; post-operative hypothermia;post-anaesthetic shivering; fatigue; tiredness; depression; cognitivedysfunction; and to enhance cognitive function.

Disorders Treated—Sensory Discomfort Etc.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of sensory discomfort.

The term “sensory discomfort”, as used herein, relates to irritation,itch, pain, or other dysesthesias (abnormal sensations; such as burningsensations, or feeling the presence of a foreign body, or pins andneedles) from the body surfaces. The term implies activation ofnociceptors located on sensory nerve endings of the body. Nociceptorsare stimulated, for example, by high or low temperatures, mechanicalpressure, chemicals (e.g., capsaicin, acidity, pollutants, etc.),injury, inflammation, and inflammatory mediators. A compound, such asDIPA-1-7, that decreases sensory discomfort, can be termed ananti-nociceptive agent.

In one embodiment, the sensory discomfort is irritation, itch, or pain.

In one embodiment, the sensory discomfort is caused by a skindysesthesia.

In one embodiment, the skin dysesthesia is skin irritation, itchy skin,or painful skin.

In one embodiment, the sensory discomfort is caused by dermatitis.

In one embodiment, the sensory discomfort is caused by atopicdermatitis.

In one embodiment, the sensory discomfort is caused by canine atopicdermatitis.

In one embodiment, the sensory discomfort is caused by psoriasis.

In one embodiment, the treatment is treatment of a skin dysesthesia.

In one embodiment, the skin dysesthesia is skin irritation, itchy skin,or painful skin.

In one embodiment, the treatment is treatment of dermatitis.

In one embodiment, the treatment is treatment of atopic dermatitis.

In one embodiment, the treatment is treatment of canine atopicdermatitis.

In one embodiment, the treatment is treatment of psoriasis.

In one embodiment, the treatment is treatment of ocular discomfort.

In one embodiment, the ocular discomfort is caused by eye strain; eyefatigue; eye surgery; an airborne irritant or pollutant that interactswith the eye surface; extended wear of contact lenses; excessiveexposure to the sun; conjunctivitis; or the dry eyes syndrome.

In one embodiment, the treatment is treatment of heat discomfort.

In one embodiment, the treatment is treatment of heat discomfort for thepurpose of improving athletic performance.

In one embodiment, the treatment is treatment of heat stress.

In one embodiment, the treatment is treatment of flushing and/or nightsweats (vasomotor symptoms) in a post-menopausal woman.

In one embodiment, the treatment is treatment of post-operativehypothermia or post-anaesthetic shivering.

In one embodiment, the treatment is treatment is to convey a sense ofrefreshment to the skin in a human.

Disorders Treated—Fatigue Etc.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of fatigue, tiredness, or depression.

In one embodiment, the treatment is treatment of fatigue.

In one embodiment, the fatigue is fatigue caused by chronic illness,ageing, a neurological dysfunction, or a psychological dysfunction.

In one embodiment, the fatigue is fatigue caused by cancer orcancer-related treatment.

In one embodiment, the fatigue is fatigue caused by anxiety, depression,heat stress, cognitive dysfunction, excessive physical exertion, orexcessive mental exertion.

In one embodiment, the fatigue is fatigue associated with a decreasedability to think, to concentrate, to study, or to perform work.

Disorders Treated—Cognitive Dysfunction Etc.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of cognitive dysfunction.

In one embodiment, the treatment is treatment to enhance cognitivefunction (e.g., in the healthy as well as the sick).

In one embodiment, the enhanced cognitive function is improved hand-eyecoordination in a sport.

In one embodiment, the enhanced cognitive function is improvedperformance in a game of chance or of mental skills.

Treatment

The term “treatment,” as used herein in the context of treating adisorder, pertains generally to treatment of a human or an animal (e.g.,in veterinary applications), in which some desired therapeutic effect isachieved, for example, the inhibition of the progress of the disorder,and includes a reduction in the rate of progress, a halt in the rate ofprogress, alleviation of symptoms of the disorder, amelioration of thedisorder, and cure of the disorder. Treatment as a prophylactic measure(i.e., prophylaxis) is also included. For example, use with patients whohave not yet developed the disorder, but who are at risk of developingthe disorder, is encompassed by the term “treatment.” Treatment toenhance the basal levels of cognitive or physical performance ofindividuals who are considered normal or healthy is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound, or a material, composition or dosage formcomprising a compound, which is effective for producing some desiredtherapeutic effect, commensurate with a reasonable benefit/risk ratio,when administered in accordance with a desired treatment regimen.

Combination Therapies

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. For example, the compounds describedherein may also be used in combination therapies, e.g., in conjunctionwith other agents.

One aspect of the present invention pertains to a DIPA compound asdescribed herein, in combination with one or more (e.g., 1, 2, 3, 4,etc.) additional therapeutic agents. The particular combination would beat the discretion of the physician or the pharmacist who would selectdosages using his common general knowledge and dosing regimens known toa skilled practitioner.

Examples of additional therapeutic agents include: an anti-inflammatoryglucocorticosteroid; an analgesic; a sympathomimetic amine decongestant;an anti-histamine; a local anesthetic; an ophthalmic lubricant; asunscreen ingredient; an anti-acne agent; a keratolytic agent; ananti-hemorrhoidal agent; an agent for vulvar itch or discomfort; anantibiotic; a skin moisturizer; or an anti-skin ageing agent.

Kits

One aspect of the invention pertains to a kit comprising (a) a DIPAcompound as described herein, or a composition comprising a DIPAcompound as described herein, e.g., preferably provided in a suitablecontainer and/or with suitable packaging; and (b) instructions for use,e.g., written instructions on how to administer the compound orcomposition.

The written instructions may also include a list of indications forwhich the active ingredient is a suitable treatment.

The written instructions (e.g., pamphlet or package label) may includethe dosage and administration instructions, details of the formulation'scomposition, the clinical pharmacology, drug resistance,pharmacokinetics, absorption, bioavailability, and contraindications.

Methods of Diagnosis

The DIPA compounds described herein may also be used in diagnosis, forexample, diagnosis of allodynia, for example, cold allodynia. Morespecifically, the DIPA compounds may be used as diagnostic agents forthe diagnosis (e.g., differential diagnosis) of cold allodynia.

Allodynia is pain due to a stimulus which does not normally provokepain. For example, temperature and physical stimuli can provokeallodynia, and it often occurs after injury to a site.

A simple diagnostic tool for differentiating neuropathic pain (e.g.,allodynia) from somatic pain is not yet known. A DIPA compound, such asDIPA-1-7, applied to the skin, can be used to provide differentialdiagnosis of, e.g., cold allodynia.

Routes of Administration

The DIPA compound or pharmaceutical composition comprising the DIPAcompound may suitably be administered to a subject topically, forexample, as described herein.

The term “topical application”, as used herein, refers to delivery ontosurfaces of the body in contact with air, which includes the skin, theanogenital surfaces, the transitional epithelial surfaces of the orbit,the lips, the nose, and the anus, and the aerodigestive tract (nasalmembranes, oral cavity, pharyngeal and esophageal surfaces), lowerrespiratory tracts, and the lumen of the gastrointestinal tract.

Particularly preferred sites of application are the surfaces innervatedby the trigeminal and glossopharyngeal nerves which include the scalp,facial skin, periorbital skin, lips, nasal and oral cavities, and thethroat. Additional preferred sites are the surfaces of the neck, elbowsand knees, which are frequently associated with the pruritus of atopiceczema and psoriasis. Yet another preferred site is the scalp, which canbe a site of inflammation in psoriasis and seborrheic dermatitis.

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment by topical administration.

In one embodiment, the treatment is treatment by topical administrationto skin.

In one embodiment, the treatment is treatment by topical administrationto facial skin.

In one embodiment, the treatment is treatment by topical administrationto periorbital skin, eyelid skin, malar skin, forehead skin, or scalp.

In one embodiment, the treatment is treatment by topical administrationto skin surface of the orbit, frontal bone, or zygomatic.

In one embodiment, the treatment is treatment by topical administrationto skin surface of the anus and/or the male or female genitalia.

In one embodiment, the treatment is treatment by topical administrationto skin above the supraclavicular fossa or the steronomastoid muscle.

The Subject/Patient

The subject/patient may be a mammal, for example, a marsupial (e.g.,kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, amouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian(e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine(e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine(e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey(e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

In one preferred embodiment, the subject/patient is a human.

Formulations

While it is possible for a DIPA compound to be administered alone, it ispreferable to present it as a pharmaceutical formulation (e.g.,composition, preparation, medicament) comprising at least one DIPAcompound, as described herein, together with one or more otherpharmaceutically acceptable ingredients well known to those skilled inthe art, including, but not limited to, pharmaceutically acceptablecarriers, diluents, excipients, adjuvants, fillers, buffers,preservatives, anti-oxidants, lubricants, stabilisers, solubilisers,surfactants (e.g., wetting agents), masking agents, colouring agents,flavouring agents, and sweetening agents. The formulation may furthercomprise other active agents.

Thus, the present invention further provides pharmaceuticalcompositions, as described above, and methods of making pharmaceuticalcompositions, as described above. If formulated as discrete units (e.g.,swab, wipe, pads, towellettes, etc.), each unit contains a predeterminedamount (dosage) of the compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

Formulations may suitably be in the form of liquids, solutions (e.g.,aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups,electuaries, mouthwashes, drops, tablets (including, e.g., coatedtablets), granules, powders, lozenges, pastilles, capsules (including,e.g., hard and soft gelatin capsules), cachets, pills, ampoules,boluses, suppositories, pessaries, tinctures, gels, pastes, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols.

Additionally, the DIPA compound may be used as an adjunct in apharmaceutical formulation or cosmetic formulation.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the DIPA compounds, and compositions comprising the DIPAcompounds, can vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects. The selecteddosage level will depend on a variety of factors including, but notlimited to, the activity of the particular DIPA compound, the route ofadministration, the time of administration, the duration of thetreatment, other drugs, compounds, and/or materials used in combination,the severity of the disorder, and the species, sex, age, weight,condition, general health, and prior medical history of the patient. Theamount of DIPA compound and route of administration will ultimately beat the discretion of the physician, pharmacist, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

Targets for Delivery

By experiment, it was discovered that the optimal targets for topicaldelivery of an agent to counteract fatigue and achieve maximal sensoryeffects are located on the receptive fields of ophthalmic and maxillarybranches of the trigeminal nerve. The preferred sites on the face areperiorbital≥zygomatic=infraorbital, labelled (f), (c), and (a),respectively, in FIG. 1. The periorbital site labelled (f) includes theskin of the eyelids and the eyelashes.

FIG. 1 is an illustration of a human head, showing facial sites fortesting:

(a) infraorbital, (b) buccal cheek, (c) zygomatic, (d)parotid-masseteric cheek, (e) frontal, and (f) periorbital. Taken fromPilsl et al., 2012.

To counteract fatigue or heat stress, the active ingredient ispreferably delivered to (a), (c), or (f). Alternatively, if the coolingagent is to be used for flushing and/or night sweats (vasomotorsymptoms) in post-menopausal women, it may also be applied to the skinabove the supraclavicular fossa or the chest. To reduce sensorydiscomfort on the skin, the cooling agent may be directly applied to thesites of injury and/or inflammation.

Secondary sites are the skin over the frontal bone and the scalp(labelled (e)), but higher concentrations of cooling agent are requiredfor (e). The other skin sites, namely, buccal cheek, parotid-massetercheek, periauricular, and chin, lack sensitivity, and sites such as thephiltrum, nasal, temporal region, and neck are topographicallyinconvenient for cooling agent delivery. In practice, the cooling agentcan be sprayed or applied (e.g., with a swab or pad or within a lotion,cream or ointment) over the skin of the orbit, the cheekbone(zygomatic), or on the skin beneath the eye, between the cheekbone andnose. The important receptive fields are from the sub-divisions of thetrigeminal nerve, namely, the zygomaticfacial nerve of the maxillarynerve (V2) and the supraorbital and supratrochlear branches of thefrontal nerve (V1).

One unusual feature of DIPA-1-7 and DIPA-1-8 is that they leave areservoir in the skin after application, so that after the initialsensations have dissipated, the dynamic cooling sensation returns whenthe skin is moist again. This feature is especially beneficial for useof DIPA-1-7 and DIPA-1-8 in conditions of elevated environmentaltemperature. When sweating is activated by heat, the sweatre-solubilizes DIPA-1-7 and DIPA-1-8 and enhances and perpetuates thesensory effect. This self-regulating feedback mechanism makes the effectof DIPA-1-7 and DIPA-1-8 more robust, efficacious, and prolonged.

Methods of Delivery

The delivery of the DIPA compounds can be achieved with the compounddissolved in a solid or semi-solid vehicle, e.g., a cream or anointment, or in a liquid vehicle, e.g., in a solution, a lotion, on aswab, wet wipe, or as an aerosolized mist.

For a solid or semi-solid vehicle, a preferred concentration of the DIPAcompound is 0.01 to 2.0% wt/vol. Unless otherwise stated, wt/vol ismeasured in units of g/cm3, and so 0.01% wt/vol is obtained from 0.1 mg(0.0001 g) DIPA compound in 1 cm3 of composition; and 2% wt/vol isobtained from 20 mg (0.02 g) DIPA compound in 1 cm3 of composition.

For a liquid vehicle, a preferred delivered volume is 0.05 to 0.15 mL.Such a volume, delivered for example as a spray, does not cause muchwetness or residue at the delivery site.

For a liquid vehicle, a preferred concentration of the DIPA compound isin the range of 0.5 to 20 mg/mL. For the orbit, a preferredconcentration is 1 to 5 mg/mL. For the zygomatic and infraorbital skin,a preferred concentration is 5 to 10 mg/mL. For the forehead skin andscalp, a preferred concentration is 10 to 20 mg/mL.

A preferred amount of the DIPA compound delivered at the site ofapplication is 0.01 to 5 mg; for example, 0.1 to 5 mg.

Wiping of the DIPA compound on the target skin can be done withpre-medicated wipes, which are well-known in personal care products, forexample, to wipe a baby's skin after a diaper change, or to removemake-up on the face (e.g., Pond's 6″×8″ (15 cm×20 cm) Clean SweepCleansing and Make-up Remover Towelettes). Usually, these wipes arepackaged as a single-use sealed unit or in a multi-unit dispenser. Forsingle units, suitable wrapper materials are those which are relativelyvapor impermeable, to prevent drying out of the wipe, and able to form a“peelable” seal. Examples of suitable wipe materials for practicing thisdiscovery include polyamide (20% Nylon)-polyester, rayon (70%)-polyester(30%) formed fabric, polypropylene nonwoven, polyethylene terephthalate(PET), polyester polypropylene blends, cotton, or microfibers (syntheticfibers that measure less than one denier or one decitex).

Alternatively, a solution containing a DIPA compound may be supplied ina reservoir bottle with individual applicators, or as a pre-packagedindividual unit. For example, Puritan 803-PCL applicators are idealcotton-tipped applicators attached to a 3-inch (^(˜)7.5 cm) polystyrenerod for delivery of a DIPA compound onto the periorbital skin. Examplesof how such applicators can be individually packaged are the SwabDose™from Unicep Corporation (1702 Industrial Drive, Sandpoint, Id., USA),and the Pro-Swabs from American Empire Manufacturing (3828 HawthorneCourt, Waukegan, Ill., USA). Each applicator tip is saturated by dippingthe absorbent material of the tip (e.g., 40 to 100 mg of cotton) in 0.5to 1.5 mL of an aqueous solution of a DIPA compound and packaged in anindividual container.

For application to the face, the individual is instructed to gentlyapply the cream, lotion, or wet wipe onto, or to spray, the targetfacial skin with the eyelids shut, or other skin surface(s). Theinstructions for application may include teaching the individual torepeat application, or “topping up”, to ensure that sufficientcomposition is delivered to the target. Once the subject has learnedwhat to expect, the individual can adjust the dosage (e.g., by dabbingat the medial or lateral edges of the orbit), as needed, to achieve thedesired effect. It has been observed that individuals learn how toeffectively apply the cooling agent after one or two trials and do sowithout risks of discomfort (e.g., eye discomfort).

For application to the anogenital skin or other highly sensitivesurfaces, the DIPA compound may be sprayed with a hand-activated manualpump, for example, to deliver volumes of approximately 0.15 mL peractivation.

Mechanisms of Action

DIPA-1-7 and DIPA-1-8 produce an anti-fatigue effect and provide reliefof heat stress and skin discomfort by evoking a sense of “dynamic cool”at sites of application. The sensation is not a steady cool, cold, oricy-cold sensation, but one of robust freshness, as if suddenly a fresh,cool breeze was blown on the skin (e.g., on the face). This effect isintense. The neurophysiological basis for this sensation, possiblereceptor mechanisms, and the significance of dynamic cooling foranti-fatigue, anti-heat stress, and anti-pruritic actions are furtherdiscussed herein.

Neurophysiology:

Small myelinated (A6) and unmyelinated fibers (C fibers) increaseafferent firing rate when skin temperature is lowered, for example,between 35° C. and 15° C. These neuronal signals that detect heatabstraction are transmitted to the central nervous system and generateconscious perception of coolness and cold. When skin temperature israised from 35° C. and 40° C., firing rates are increased in C fibersand these fibers signal warmth (see, e.g., Hutchison et al., 1997). Thereceptive mechanisms and “cable lines” for cool/cold and warm areseparate and distinct, but reciprocally inhibit each other in the brainand perhaps also in the periphery. The sensory receptors are modalityspecific and do not respond to mechanical stimulation. At the molecularlevel, the target binding sites for cooling agents are thought to belocated on ion channel receptors that depolarize in response to a dropin temperature. Heat abstraction decreases the threshold for dischargeof the receptor, and the facilitated depolarization initiates the axonalresponses that create the neuronal signal.

The central response of these neurons has been recorded and studied fromrat superficial medullar dorsal horn that responds to innocuous thermalstimulation of the rat's face and tongue. Step changes of −Δ5° C.stimulated cells with both static firing rates and cells that had mainlydynamic properties (see, e.g., Davies et al., 1985). Similar studies incats and humans showed that step decreases in temperatures (dynamicchanges), as low as Δ0.5° C./second, were readily detectable by neuronsand by psychophysical measurements (see, e.g., Davies et al., 1983).

From a study of the spike patterns of neuronal discharge(impulses/second), it was clear that dynamic, and not static firingresponses to a change in temperature were the most powerful stimuli forgenerating coolness/cold sensations (see, e.g., Hutchison et al., 1997).That is, the brain “sees” −Δ° C./t and not absolute ° C. Thus, a coolingagent that simulates −Δ° C./t on nerve discharge will produce “dynamiccooling”.

Relationship of Dynamic Cooling to Anti-Fatigue:

Dynamic cooling (versus static cooling/cold) is essential for ananti-fatigue effect. For example, if one is tired and driving a vehicle,turning on the air-conditioning and blasting the air onto the face willcounteract fatigue. But just turning on the air conditioner to lowerambient temperature and being chilled inside the vehicle will not makemuch of a difference.

The topical therapy for enhanced performance and counteract fatiguedescribed herein circumvents the necessity for systemic drugs that actinvasively on brain chemistry. The benefits of the topical therapy areillustrated by the Case Studies described herein.

Receptor Mechanisms:

There is a general view that “TRP-” ion channel receptors (A1, M8, andV1 to 4) are the principal physiological elements for physiologicaltemperature detection. The TRPM8 receptor is the one that responds tosensory/cooling agents such as menthol and icilin (see, e.g., McKemy,2002). TRPM8 is a protein with 1104-amino acid residues and has sixtransmembrane domains. Activation of this receptor by lowering ambienttemperature results in opening a pore between the 5th and 6thtransmembrane loop and non-specific cation entry into the cell.Depolarization of TRPM8 receptors on sensory neurons may then transmitsignals primarily via Aδ (and some C) fibres.

While this concept for the role of TRPM8 in sensory physiology may bevalid for physical changes in temperature, the interpretation of thesensory effects of chemical agents such as menthol and icilin are morecomplex. Menthol not only stimulates TRPM8 in vitro, but also TRPV3, areceptor associated with warmth (see, e.g., Macpherson et al., 2006).Menthol also inhibits TRPA1. Icilin stimulates not only TRPM8, but alsoTRPA1, and icilin inhibits TRPV3 (see, e.g., Sherkheli et al., 2012) andglycinergic transmission (see, e.g., Cho et al., 2012). Thus, mentholand icilin are “promiscuous” cooling agents and their specific sensoryeffects may not be associated with any one particular receptor protein.The Inventor has screened a large database of cooling agents but,surprisingly, only DIPA-1-6 and DIPA-1-7 produced super-robust dynamiccooling on skin. DIPA-1-8 also produces strong cooling and its actionsare prolonged, but it does not quite have the super “wow” coolingeffects of DIPA-1-6 and DIPA-1-7. Other cooling agents are lessstimulating or have shorter durations of action and thus less suitablefor the uses contemplated herein.

It may be concluded that DIPA-1-7 and DIPA-1-8 bind to a site on avoltage-gated ion channel receptor located on a nerve ending that issensitive to a decrement in physical temperature. This event facilitatesneuronal depolarization to a cooling/cold signal, and an actionpotential is transmitted via Aδ and C fibers towards the central nervoussystem. If the nerve ending is located on the facial skin, the signal isrecordable from dorsal surface of the trigeminal nucleus in thebrainstem. Further rostral transmission and integration of signals giverise to the perception of coolness/cold and its topographicalassociation with the site of stimulation.

When one examines the structure-activity relationships (SAR) of the DIPAcompounds, it is noted that when R1=R2=isopropyl and R3=n-hexyl (C6) orn-heptyl (C7), then dynamic cooling is observed. Strong cooling of longduration is also obtained with R3=n-octyl (C8). However, whenR1=R2=sec-butyl and R3=n-butyl to n-octyl (C4 to C8), dynamic cooling ispartially observed, but with much less intensity. As shown in thestudies described herein, this distinction between di-sec-butyl anddi-iso-propyl compounds is also seen in animal studies on shakingbehaviour, an indicator of cooling actions in the rat (because shakingis inhibited by heat).

Shaking behaviour is a rapid alternating contraction of the supinationand pronation muscles about the spinal axis, and can be readily observedand counted. Fur-coated and feathered animals—when wet and cold—shake,like a wet dog (see, e.g., see, e.g., Dickerson et al., 2012;Ortega-Jimenez et al., 2012; Wei, 1981). “Wet-dog shaking” has beenstudied in detail in animals. Rats can shake their head, the uppertorso, or the shaking can be sufficiently violent to affect the wholebody and make the animal lose its balance. DIPA-1-7 and DIPA-1-8 elicitthe vigorous type of shaking. The purpose or survival value of shakingto fur-coated and feathered organisms is to remove water dropletstrapped on or near the skin. Removal of the water droplets on or nearthe skin by shaking reduces the organism's need to expend energy toremove the water by evaporation. The likely equivalent behaviour toshaking in humans is shivering, a condition caused by generalizedsensations of coolness/cold. Human subjects recovering from the deephypothermia of anaesthesia manifest vigorous shaking; a condition calledpost-anaesthetic shivering.

Icilin(1-[2-hydroxy]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one)induces vigorous shaking in rats. Surprisingly, two potent p-menthanecarboxamide cooling agents[(R)-2-[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-propionicacid ethyl ester,[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-aceticacid isopropyl ester], which have EC50 values similar to icilin at theTRPM8 receptor, do not evoke shaking (when injected at 50 mg/kg s.c. inmale rats and observed for 1 hour). Icilin activation at the TRPM8receptor is abrogated by a G805A mutation at the second to thirdtransmembrane loop, but the effects of menthol are not affected. It islikely that DIPA-1-6, DIPA-1-7, and DIPA-1-8 also have specific sites ofbinding and activation on the TRPM8 receptor which are not shared bymenthol or p-menthane carboxamides, but recent studies have shown thatDIPA-1-6 and DIPA-1-7 are still active on TRPM8 receptors with the G805Amutation.

The studies described in Watson et al., 1978, show that the presence ofa polar oxygen moiety capable of acting as an acceptor of a hydrogenbond from the receptor is essential for bioactivity. A Hückel molecularorbital calculation (using Molecular Modelling Pro v6.0.3, ChemSW Inc,Fairfield, Calif. 94534, USA) on the isopropyl analogs versus thesec-butyl analogues favours a slightly higher partial negative charge(0.007e) on the oxygen in the sec-butyl entities, suggesting that thesec-butyl substituents facilitate a higher affinity of the oxygen to thehydrogen binding site of the receptor. Thus it is possible thatisopropyl, with a “looser” affinity can associate and disassociate withthe receptor more rapidly, favouring the generation of a dynamic onsetand offset response of the receptor. This rapid interaction with thebinding site will favour a more “dynamic” and intense stimulation ofcooling and give rise to the phenomenon known as shaking.

Another possibility is that DIPA-1-7 has a dual action on TRP receptors,so that it stimulates TRPM8 and, at higher concentrations, stimulatesTRPV1. The dual action will give a cold-hot synergy that might lead to amore dynamic cooling sensation.

TRPM8, TRPA1, and TRPV1 Receptor Assays:

The in vitro effects of test compounds were evaluated on cloned hTRPM8channel (encoded by the human TRPM8 gene, expressed in CHO cells) usinga Fluo-8 calcium kit and a Fluorescence Imaging Plate Reader(FLIPRTETRA™) instrument. To examine the specificity of the testcompounds, further tests were conducted on TRPV1 channels (human TRPV1gene expressed in HEK293 cells) and TRPA1 channels (human TRPA1 geneexpressed in CHO cells). The assays were conducted by ChanTestCorporation, 14656 Neo Parkway, Cleveland, Ohio 44128, USA.

Selection of Active Ingredient

Ideally, an active pharmaceutical ingredient (API) formulated fordelivery to the keratinized skin should be stable, non-toxic, andsufficiently long-acting and potent to activate the mechanisms thatresult in an anti-fatigue, anti-heat, or anti-nociceptive effect. TheAPI should be dissolved and evenly dispersed in a composition so thatduring manufacture the formulation maintains a constant concentration.The final product should meet standards of cleanliness and sterility.For purposes of formulation, the API can be a liquid at standardconditions of temperature and pressure (STP) and that is evenlydissolved in aqueous solutions at neutral pH and/or isotonicity.Sterility of the final product can be optimally achieved by usingpurified reagents and filtration through micropore filters, heating, orirradiation. Standard excipients, such as emulsifying agents, isotonicsaline, solvents, stabilizing agents, and preservatives, may be added tooptimize the formulations, but the important ingredients should bepreferably soluble in aqueous media such as purified water or a standarddermatological solvent.

For a given individual, the perceived sensation is a function of theparticular cooling agent, the dose, the vehicle used to carry thecooling agent, the method of topical delivery, and the nature of thetarget surfaces. The Inventor has screened a number of candidatecompounds on the facial skin (see, e.g., Wei, 2011) and has identifiedDIPA-1-6, DIPA-1-7, and DIPA-1-8 as having the preferred desiredproperties of an ideal anti-fatigue, anti-heat, and anti-nociceptiveagent.

To summarize, the concepts that led to DIPA-1-6, DIPA-1-7, and DIPA-1-8as being suitable agents are:

-   -   The definition of a rationale for using a “dynamic cool”        sensation around the orbit and zygomatic to combat fatigue, and        describing the neurophysiology and mechanisms of this action.        This sensory effect in unusual and found in the DIPA compounds        but not found with structurally similar compounds.    -   Devising a delivery method for the API which avoids contacting        nociceptors on the cornea because that will result in sting/pain        and be aversive and not practical.    -   Finding an ideal compound (API) by experiment: DIPA-1-7 and        DIPA-1-8 are water soluble (a clear solution is obtained at up        to 20 mg/mL in distilled water), stable to heat, and exerts a        “dynamic cool” sensation for five to seven hours at an applied        concentration of 1 to 10 mg/mL. Tachyphylaxis does not develop        to repeat applications.    -   Defining the receptor targets of these compounds in vitro, and        the selectivity of the chosen API.    -   Defining an in vitro isolated nerve preparation that shows an        anti-nociceptive action of DIPA-1-7, and showing that this        effect is abrogated on the nerve from a TRPM8 knockout mouse.    -   Defining an animal model (of “wet-dog shakes”) that will        illustrate the “dynamic cool” properties and allow further study        of mechanisms of action.    -   Conducting tests in human volunteers that show efficacy of the        DIPA compounds for reducing fatigue caused by chronic illness        and heat stress, and for increasing mental energy levels in the        normal person.    -   Conducting tests in human volunteers that show DIPA-1-7 is        effective for relieving sensory discomfort of the skin, and thus        may be used as an anti-nociceptive or anti-pruritic agent, or as        a diagnostic tool for evaluating skin dysesthesias.

Applications

The DIPA compounds, when applied to keratinized skin, havesensory/cooling effects that mimic heat abstraction, but without achange in tissue temperatures. These compounds, especially DIPA-1-6,DIPA-1-7 and DIPA-1-8, can also penetrate the skin barrier and enter thesystemic circulation to exert a cooling action. These effects areobtained at small volumes, e.g., 0.1 to 0.5 mL, applied at aconcentration of 1 to 20 mg/mL, or 0.1 to 2% wt/vol. The onset of effectis rapid, less than 5 minutes, and the sense of coolness is robust,refreshing, and strong. Compounds with similar bioactivity on thekeratinized skin are not currently used in cosmetic or therapeuticapplications.

Heat Stress:

Thermal comfort is a technical term used by air-conditioning engineersto define “a state of mind in humans that expresses satisfaction withthe surrounding environment.” Maintaining thermal comfort for occupantsof buildings or other enclosures is one of the important goals ofarchitects and design engineers. For most people, the room temperaturefor thermal comfort is 25° C. (77° F.). Careful studies have documentedthat work performance and productivity (output/input) drop by 2% forevery increment of +1° C. above 25° C. up to 33° C. At officetemperatures of 28-30° C. (82-86° F.), there is increased sweating andcomplaints of headache, drowsiness and dullness, difficulty inconcentrating, and physical discomfort. For example, studies have shownthat increasing the indoor air temperature of a call center from 25° C.to 26° C. decreased the call response rate from 7.79 to 7.64 calls/hr, a1.9% loss (see, e.g., Tanabe et al., 2007). An ambient temperature above25° C. is thus a form of heat stress.

Energy consumption of buildings in China account for at leastone-quarter of the country's energy use, and sales of air-conditioningsystems in Brazil and India are on an exponential increase. This rise inenergy use has raised further concerns about global warming, but as mostpopulations now work indoors, energy costs must be balanced againstworker productivity. Basically, a worker's efficiency is better when heor she is kept cool. A method for combating mental lassitude from a hotenvironment, without incurring expenditures for energy, would haveeconomic benefits. In the Case Studies describe herein, it was foundthat application of DIPA-1-7 to the facial skin of a student, preparingfor exams, was useful in overcoming the discomforts of heat.

Athletic Performance:

It is a natural desire of humans to want to perform better, eitherphysically or mentally. Recently, there has been an enthusiastic surgeof interest in the use of cryotherapy to improve athletic performance.Cryotherapy is defined as “ . . . the lowering of tissue temperature(locally or generally) by the withdrawal of heat from the body toachieve a therapeutic objective . . . ” It is now accepted that externalpre-cooling by heat abstraction, for example, by immersion in ice or bywearing a vest packed with ice, can improve work endurance in a hotenvironment (see, e.g., Marino et al., 2002). An increase in physicalwork output of ^(˜)5% can be shown for tasks of approximately 30 minutes(see, e.g., Grahn et al., 2005). Heat exhaustion limits work and thisoccurs when core body temperature approaches 40° C. (104° F.).Pre-cooling (or internal cooling, for example, by drinking an iceslurry) slows down the rate of heat accumulation.

Surprisingly, the improvement in athletic performance can be attained bythe perception of coolness, without modifying core temperature.Investigators showed that trained marathon runners wearing a commercialcooling collar (Black Ice LLC, Lakeland Tenn.) extended the time toreach volitional exhaustion by 13.5% (see, e.g., Tyler et al., 2011).Cooling of the neck dampened the perceived level of thermal strain anddelayed the point of voluntary termination of exercise. Participantstolerated a higher body temperature and heart rate when their neckregions were cooled.

In several studies with menthol, a chemical that produces sensations ofcoolness without a change in skin or core temperatures, it was noticedthat an increased perception of cooling, without a change in core bodytemperature, may also enhance better physical performance. This effectwas unexpected and attributed to menthol being a “positive” placebo(see, e.g., Gillis et al., 2010; Schlader et al., 2011). The surface ofthe face is densely innervated with nerve endings that detecttemperature. The peripheral cool/cold detection system is associatedwith specific nerve fiber discharges and precisely regulated so ±1° C.is easily discriminated. Over 92% of thermoceptive units on the face,especially around the lips, respond to cooling and these neurons aretonically active at room temperature (see, e.g., Hutchison et al.,1997).

Menthol causes irritation when it is sprayed on the skin at 2% or more.Exercise performance is not enhanced after spraying, although subjectsreport a cooling sensation. It is likely that an agent such as DIPA-1-7or DIPA-1-8, applied to the face, neck region, or chest will decreaseheat discomfort and improve athletic performance.

Illness-Related Fatigue:

Fatigue is recognized as an important problem for patients with advancedprogressive illness, especially cancer, as fatigue negatively affectsphysical, psychological, social and spiritual well-being, and quality oflife (QOL) (see, e.g., Minton et al., 2010). This symptom is identifiedas a condition that requires management and research priority. Forcancer-related fatigue: a consensus definition is “a common, persistent,and subjective sense of tiredness related to cancer or to treatment forcancer that interferes with usual functioning”.

Assessment instruments specific for fatigue have been developed such asthe Brief Fatigue Inventory, the Cancer Fatigue Scale, the FatigueAssessment Instrument, and the Multidimensional Fatigue Inventory. Theimportant questions asked of patients are:

(1) Do you feel or have you ever felt unusually tired? (2) If yes, canyou indicate how tired you feel on average on a scale from 0 to 10? (3)How much does this tiredness affect your daily life activities?

Related symptoms of fatigue are: complaints of generalized weakness orlimb heaviness, diminished concentration or attention, diminishedenergy, increased need to rest, decreased interest in engaging in usualactivities, insomnia or hypersomnia, experience of sleep asun-refreshing or non-restorative, difficulty in completing daily tasksattributed to feeling tired, perceived problems with short-term memory,and changes in emotional reactivity (e.g., sadness, frustration, orirritability). If five or more these symptoms are present every day ornearly every day during a 2-week period, then a diagnosis of medicalfatigue is made.

Using these questionnaires it has been estimated that fatigue is presentat the time of diagnosis in approximately 50% of cancer patients, andcan increase to 60-96% of cancer patients during treatment.

In addition to cancer, other serious illnesses in which fatigue has beenexamined for interventions include chronic obstructive pulmonarydisease, motor neuron disease, cystic fibrosis, dementia, Parkinson'sdisease, human immunodeficiency virus/acquired immune deficiencysyndrome, and multiple sclerosis. Recognised potential causes of fatigueinclude anemia, dehydration, infection, malnutrition, pain, depression,disturbed sleep, anxiety, hypothyroidism, disease progression, andmuscle wasting and deconditioning. A feature of fatigue in thesepatients includes feeling tired without exertion and even after resting.Patients complain of a reduced capacity to carry out the normalactivities of daily living, slow physical recovery from tasks, anddiminished concentration.

Management of fatigue includes drugs such as antidepressants,analgesics, stimulants, anxiolytics and nutritional supplements.Non-drug methods include counseling on improved sleep practices,physical exercises, and relaxation techniques. Erythropoietin anddarbepoetin, drugs that stimulate red blood cell production, areeffective, but may decrease survival, and this adverse effect limitstheir use. In reviews of the literature, no drugs that work as centralnervous stimulants other than methylphenidate exhibit clearly identifiedbenefits to counter fatigue (see, e.g., Payne et al., 2012). Fatigue isconsidered a condition that requires research priority because otheradverse effects of cancer treatment, namely, pain and nausea, arerelatively well-managed, but fatigue is not.

Topical application of a “dynamic cool” agent such as DIPA-1-7 orDIPA-1-8 may have utility to counter-act fatigue, refresh, and toinvigorate.

Cognitive Enhancement:

It is a natural desire of humans to want to perform better, eitherphysically or mentally. Chemicals designed to enhance performance belongto two categories: those that increase physical capabilities, e.g.,anabolic steroids or vitamins, and those that increase cognitivefunctions. Drugs that are “cognitive enhancers” (CEs) are also callednootropic drugs or neuroenhancers, and include substances such ascaffeine, amphetamines, methylphenidate, nicotine, donepezil, andmodafinil. The CEs are designed to enhance the individual's capacity fortasks such as abstract thinking, attention, attitude, brainstorming,comprehension, recognition, creative thinking, critical thinking,increasing curiosity, executive functions, decision making, eideticmemory, emotions and feelings, goals and goal setting, imagination,intelligence, introspection, lateral thinking, learning, memory, mentalcalculation, motivation, perception, personality and recollection(recall).

Conscious perception of the visual world depends on the visual system tocapture image patterns on the retina and to deliver it to the brain forcognition and understanding. Cognitive functioning is the sum of memory,intelligence, creativity and attention. Human attention is furtherdivided into attentional tone (the state of vigilance) and selectiveattention (the ability to focus on and to execute a task without beingdistracted). The brain network for attention and its pharmacology hasbeen the subject of reviews (see, e.g., Lanni et al., 2008). Theneurotransmitter mechanisms of some CEs have been investigated. Drugssuch as amphetamines and methylphenidate increase vigilance viacatecholaminergic pathways and nicotine and donepizil may affectselective attention via cholinergic pathways. The visual system isespecially important to an organism's survival and it is estimated byneurophysiologists that at least 90% of the organism's brain activity isfocused on processing and interpreting visual sensory input.

Not all chemicals that affect brain/behavior enhance performance. Forexample, alcohol (ethanol) and cannabis are not cognitive enhancers. Adecrement in cognitive performance is called cognitive dysfunction (orimpairment) and can be manifested as fatigue, sleepiness, loss ofmemory, and inability to learn, to make decisions, to complete tasks, orto follow instructions. Cognitive dysfunction leads to decreased jobproductivity, transportation system accidents, inability to perform, anddaytime fatigue/sleepiness. Many conditions can lead to cognitivedysfunction and impairment including ageing, anxiety, depression,Alzheimer's disease, strokes, Parkinson's disease, narcolepsy, insomnia,disruption of circadian rhythms, obstructive sleep apnea, anddepression.

Use of drugs such as CEs in the healthy, e.g., in the academic andbusiness environment, has been the subject of much recent debate (see,e.g., Talbot, 2009; Greely, 2008). Currently, the drugs used requireaccess of the active agent into the bloodstream, and onto centralnervous system enzymes or receptors. Here the proposed method of CE isachieved by topical administration of an agent with a “dynamic cool”effect onto the external surface of facial skin and there is no directinvasion of brain chemistry.

It may be asked why cognitive functions should be enhanced by a DIPAcompound. If you ask a person from a cold climate (e.g., Norway, Russia,or Korea) if frigid air on the face will wake you up and think moreclearly, they will state that this in a known experience and an obviousfact. Frigid cold weather makes people think more clearly. The dynamiccool produced by DIPA-1-7 is a similar alerting event.

Without wishing to be bound by any particular theory, the Inventorproposes the following hypothesis as an explanation for this phenomenon.Approximately 200 million years ago certain organisms acquired theability to control metabolic heat production (endothermy) and tomaintain a constant internal body temperature (homeothermy). Thisevolutionary transition, from a “cold-blooded” to a “warm-blooded”physiology, enabled such species to better adapt and to survive in avariable environment. Although humans primarily evolved in a warmhabitat, migration has also exposed the species to cold. Coolness is thefirst signal to warn of the need for heat conservation and is apervasive and dominant neuronal signal for ensuring the organism'ssurvival because the metabolic machinery of the organism operatesefficiently at, and is dependent on, a constant temperature. In thepresence of cold, an organism thinks and plan for survival. Thiscircuitry is built into the brain, and serves as a template forenhancement of cognitive function.

Sensory Discomfort from Body Surfaces:

The potent “dynamic cool” sensations produced by DIPA-1-7 and DIPA-1-8were further evaluated for anti-itch (and other anti-nociceptive)effects on skin. As shown in the Case Studies described herein, a 20mg/mL solution, applied with a cotton-tipped applicator potently stoppeditching and discomfort caused by contact dermatitis in threeindividuals.

A topical medication that can relieve sensory discomfort has manyapplications including:

(a) alleviation of irritation, itch and pain from various forms ofdermatitis (atopic, contact, and irritant);

(b) pain from burned, traumatized, diseased, anoxic, or irritated skin(e.g., skin damaged by laser surgery, diabetic ulcers, sunburn,radiation), and from procedures related to wound debridement;

(c) itch and discomfort from skin infections, insect bites, sunburn,photodynamic treatment of skin (e.g., actinic keratoses, basal cellcarcinoma), lichen sclerosus;

(d) pruritus due to xerosis, psoriasis, or seborrheic dermatitis;

(e) mucositis, stomatitis, cheilitis, itching of the lips from coldsores or gingivitis;

(f) pruritus ani, hemorrhoidal discomfort, pain from anal fissures, painor itch from anal fistulas, pain from hemorrhoidectomy, perinealinflammation, anogenital skin inflammation and discomfort due to variouslocal causes such as incontinence, diaper rashes, perineal inflammation;

(g) vulval pruritus and pain (e.g., from candidiasis or idiopathic, suchas vulva vestibulitis and vulvodynia), dyspareunia, anogenitalinfections, including warts and sexually transmitted diseases, fungalinfections, viral infections of the skin (especially inimmunocompromised patients);

(h) nostril and nasal or upper airway discomfort from breathingobstruction, e.g., congestion, rhinitis, asthma, bronchitis, emphysemaand chronic obstructive pulmonary diseases, dyspnea, sleep apnea andsnoring; and

(i) conjunctivitis, ocular surface irritation, pain from cornealabrasions, and pain from eye surgery.

Of special interest, is the use of DIPA-1-7 and DIPA-1-8 for scalp itch,e.g., in seborrheic dermatitis and psoriasis; these end-points beingunmet medical needs. DIPA-1-7 may also be used to refresh the skinbefore application, or after removal of, cosmetics from the skin, toreduce the irritant effects of benzyoyl peroxide in the treatment ofacne, and to reduce sebum secretion and the appearance of an “oily”skin.

Vasomotor Symptoms (“Hot Flushes/Night Sweats” in Post-MenopausalWomen):

Flushing (vasodilation) and sweating occurs on the body when the brain'sthermoregulatory system perceives a need to lower body temperature.After menopause, at least one-third of women experience “hot flushes”(i.e., brief but repetitive episodes of feeling warm and flushed, anddaytime and nighttime sweating). Replacement estrogens may alleviatesymptoms but there are uncertainties if hormone replacement therapy(HRT) is safe. Sweating episodes that occur at night and in the earlymorning hours are especially inconvenient because the bed-sheets becomewet and it is burdensome to change the bed-sheets on a daily or frequentbasis. Episodes of “hot flushes/night sweats” can occur as often as onaverage 14 episodes per week. Aside from HRT, current alternativemethods of therapy, such as yoga, acupuncture and phytoestrogens, havenot been shown to be effective.

The DIPA compounds are potent agents that can cross the skin barrier andbe absorbed into the bloodstream and exert systemic effects. Onepossible method of treating vasomotor symptoms may be to topicallyadminister DIPA-1-6 or DIPA-1-7 via a controlled-release patch. Thesystemic effects of the DIPA compound will then give rise to coolingsensations to counteract activation of central heat-loss mechanisms(vasodilatation and sweating). The patch may be applied at night to aconvenient location of the body, e.g., the skin above thesupraclavicular fossa or the skin above the sternomastoid muscle, andthe released DIPA compound would then inhibit the “night sweats.”Alternatively, the DIPA compound (e.g., DIPA-1-6, DIPA-1-7, or DIPA-1-8)can be applied to the skin as a cream or lotion.

Diagnostic Agent for Allodynia:

Patients with neuropathic pain frequently suffer from painful sensationsinduced by normally innocuous skin cooling, a condition called coldallodynia (see, e.g., Wasner et al., 2008). Cold allodynia is seen insome diabetic patients with pain, but a simple diagnostic tool fordifferentiating neuropathic pain from somatic pain is missing. An agentsuch as DIPA-1-7 applied to the skin may be useful for such diagnosisand aid in the selection of the best method for therapy. A 40% mentholsolution in alcohol has been used as a challenge agent, but the resultsin the clinic have been ambiguous (see, e.g., Binder et al., 2011).

Prevention of Post-Operative Hypothermia and Post-Anaesthetic Shivering:

Surgical patients with mild peri-operative hypothermia (33 to 36.4° C.)and post-anaesthetic shivering have a greater risk of adverse outcomes,including events such as decreased wound healing, increased bleeding,and morbid cardiac events (see, e.g., Buggy et al., 2000). A study hassuggested that a TRPM8 agonist such as menthol, by producing coldsensations, can elevate core temperature (see, e.g., Tajno et al.,2011). An agent such as DIPA-1-7, by increasing sensitivity to cold, maybe an useful as a drug treatment against post-operative hypothermia. Inthe rat, injection of DIPA-1-7 induces shaking, elevation of bodytemperatures, and a shortening of the duration of pentobarbitalanesthesia, as measured by recovery of the righting reflex. Thesepharmacological actions will counter the depressive effects ofanesthetics on body temperature.

Pharmaceutical Adjunct:

In pharmaceuticals or cosmeceuticals, the term “adjunct” is anadditional substance, treatment, or procedure used for increasing theefficacy or safety of the primary substance, treatment, or procedure orfor facilitating its performance. The DIPA compounds relieve sensorydiscomfort of the skin, have anti-nociceptive activity, and are activeat less than 1 minute after application. They are ideal adjuncts forpharmaceuticals and for cosmetics applied to the skin.

If the primary substance is an irritant, the adjunct may be used todecrease irritancy, and hence improve patient tolerance and compliance.For example, an adjunct such as DIPA-1-7 can be added an anti-acnepreparation containing benzoyl peroxide. Benzoyl peroxide, the primarysubstance, works as a skin peeling agent, increases cell turnover, andreduces P. acnes, but it is an irritant and can cause burning, swelling,and pain when applied to the skin. Similarly, imiquimod (Aldara®), whichis used as a primary substance to treat genital warts and skin cancercan cause blistering and pain, and an adjunct such as DIPA-1-7 orDIPA-1-8 may increase patient acceptance and compliance in the use ofthis drug.

An adjunct such as DIPA-1-7 may be used to increase the “apparent”efficacy of another primary ingredient, and thereby improve patientsatisfaction and adherence to a dosage schedule. For example, DIPA-1-7at about 0.5 to 2%, stops itching within minutes after application. Ifcombined with an anti-inflammatory steroid, the preparation may be moredesirable than the anti-inflammatory steroid alone, which takes longerto act. Anti-inflammatory steroids, such as hydrocortisone,triamcinolone, and clobetasol are used for sensory discomfort of theskin in disorders such as insect stings, contact dermatitis, atopiceczema, and psoriaisis. The presence of DIPA-1-7 as an adjunct, inaddition to helping to stop the itch, may help reduce the dose or thefrequency of application of the primary ingredient, yet achieve anequivalent therapeutic effect. This adjunct benefit will be especiallybeneficial in the use of skin steroids because of the well-knownundesirable effects of collagen degradation, tissue thinning, andincreased susceptibility to infections. An adjunct that reduces dosageor promote greater efficacy of the primary ingredient has value. Otherprimary anti-pruritics are aluminum acetate, and strontium chloride orstrontium nitrate.

For skin disorders, compositions of the present discovery may also beused as adjuncts for procedures such as phototherapy, laser therapy,cryotherapy, or UV-therapy of the skin.

Pharmaceuticals that may be used, in combination or in sequence withadjunct DIPA compounds include anti-inflammatory steroidal agents,anti-inflammatory analgesic agents, antihistamines, sympathomimeticamine vasoconstrictors, local anesthetics, antibiotics, anti-acneagents, topical retinoids, drug for genital warts and skin cancer, drugsfor wrinkles and ageing skin, anti-hemorrhoidal agents, drugs for vulvaritch, skin moisturizers, and agents for keratolysis.

Examples of steroidal anti-inflammatory agents include hydrocortisone,clobetasol, clobetasol propionate, halobetasol, prednisolone,dexamethasone, triamcinolone acetonide, fluocinolone acetonide,fluocinonide, hydrocortisone acetate, prednisolone acetate,methylprednisolone, dexamethasone acetate, betamethasone, betamethasonevalerate, flumetasone, fluticasone, fluorometholone, beclomethasonedipropionate, etc.

Examples of anti-inflammatory analgesic agents include methylsalicylate, monoglycol salicylate, aspirin, indomethacin, diclofenac,ibuprofen, ketoprofen, naproxen, pranoprofen, fenoprofen, sulindac,fenclofenac, clidanac, flurbiprofen, fentiazac, bufexamac, piroxicam,pentazocine, etc.

Examples of antihistamines include diphenhydramine hydrochloride,diphenhydramine salicylate, diphenhydramine, chlorpheniramine maleate,promethazine hydrochloride, etc.

Examples of sympathomimetic amine vasoconstrictors include phenylephrinehydrochloride, oxymetazoline, naphazoline, and other imidazolinereceptor agonists used for nasal decongestant activity and for rednessand vasodilatation on the ocular surfaces.

Examples of local anesthetics include dibucaine hydrochloride,dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, pramoxinehydrochloride, tetracaine, tetracaine hydrochloride, oxyprocainehydrochloride, mepivacaine, piperocaine hydrochloride, etc.

Examples of skin moisturizer ingredients include the three categories ofhumectants, emollients and preservatives. Humectants, such as urea,glycerin and alpha hydroxy acids, help absorb moisture from the air andhold it in the skin. Emollients, such as lanolin, mineral oil andpetrolatum, help fill in spaces between skin cells, lubricating andsmoothing the skin. Preservatives help prevent bacteria growth inmoisturizers. Other ingredients that moisturizers may contain includevitamins, minerals, plant extracts and fragrances.

Examples of antibiotics include neomycin, erythromycin, and theanti-viral agent docosanol (Abreva®), and experimental agents such asN,N-dichloro-dimethyltaurine. Topical anti-acne agents include benzoylperoxide, resorcinol, resorcinol monoacetate, and salicylic acid. Otheragents to counter acne include topical retinoids such as adapalene andisotretinoin (Retin-A, Differen, and Tazorac). Examples of keratolyticsinclude such agents as, alpha-hydroxy acids, glycolic acid, andsalicylic acid.

The adjunct DIPA compound can be used for medications that are usefulfor human therapy as well as for veterinarian uses.

Study 1 Toxicity

Preliminary toxicological studies were conducted on DIPA 1-7. It was notmutagenic in the Ames test (Strains TA 98 and TA100, with and withoutliver activation) (tests conducted by Apredica, Watertown, Mass., USA).

DIPA-1-7, dissolved in 3% ethanol/97% 1,2-propanediol, or vehicle alone,was administered at 20 mg/kg perioral for 7 days (N=10 per group) tomale rats, and on the 8th day, the animals were euthanized with sodiumpentobarbital and the major organs (body, heart, liver, lungs, kidney,testes, brain) were removed and weighed. Heart tissues (ventricle andheart valves) and liver samples were stained with hematoxylin and eosinand the histology examined. There was no significant difference in bodyor organ weights between the two groups and the heart and liverhistology were normal.

Study 2 Tissue Temperature

The compounds of the present invention simulate the sensations of heatabstraction, but do not alter tissue temperatures. The average foreheadskin temperature of subjects (N=5) was measured following application ofDIPA-1-7 (with a wipe at a concentration of 20 mg/mL in distilled water)to the forehead skin. The results are summarized in the following table.The subjects noted the cooling effect of DIPA-1-7 on the skin whichlasted for 30-45 minutes; however, skin temperatures were not affected.

Temperature (° C.) Time Control DIPA-1-7 Before 37.3 37.4  0 minutes37.2 37.4 15 minutes 37.5 37.5 30 minutes 37.1 37.1 45 minutes 37.4 37.260 minutes 37.0 37.1

Study 3 Sensory Effects of Compounds on Facial Skin

When a test compound is applied to the skin, it is possible tocharacterize the resulting sensations. The quality of the sensationsproduced by individual compounds favours certain characteristics thatare distinct. The quality of the sensations evoked, their descriptors,and their proposed mechanism of action, are summarised in the followingtable. For any compound, there may be some overlap in activity, butusually one compound occupies only one or two categories of sensations.For example, icilin is exclusively cool, with very little “cold”.DIPA-1-6 and DIPA-1-7 are exceptional in producing pleasant, robust“dynamic cool.” DIPA-1-8, 2-6, are 2-7 are strong cold-producing agents.

Proposed Mechanisms on Type of Sensation Descriptor Sensory NeuronsInactive No effect — Cool, steady and pleasant Cool Balanced stimulationof static and dynamic Cold, constant, but limited Cold Higherstimulation of static by desensitization Dynamic cooling, robust Dynamiccool Higher stimulation of dynamic cool/cold, strong refreshing Stingingcold, sometimes Icy cold Stimulation of dynamic and with irritationstatic, and also nociceptive sites

Even after the offset of the cooling/cold action, some of the compoundshave a “reservoir effect.” Experimentally, this is measured 1 hour afteroffset by placing a hot and then a cold towel over the site ofapplication and determining if the onset of cooling/cold returns for atleast 30 minutes. If this occurs, then there is a positive “reservoireffect”. The “reservoir effect” can also be provoked with air movement,but the conditions for air movement are difficult to standardize. The“reservoir effect” of DIPA-1-7 in skin is most likely due to residualdrug that is reactivated to stimulate dynamic/static sensory neurons.

In the studies described herein, the sensation of coolness/cold is ratedas 0, 1, 2, or 3 with: 0 as no change; 1 as slight coolness, or cold; 2as clear-cut signal of coolness or cold; and 3 as strong cooling orcold. The sensations are recorded at intervals of 5 to 15 minutes, untilat least two successive zeroes are obtained.

The onset of drug action is taken as the time to reach 2 units ofcoolness intensity.

The duration of sensory action is defined as the offset time minus theonset time. The offset of drug action is defined here as the time whencoolness intensity drops below 2, after previously surpassing 2 units.An inactive compound is defined as one that does not exceed 2 units ofcooling for 5 minutes or more after application. The offset endpoint issometimes unstable for compounds that act for two or more hours, becausethe coolness/cold sensation may fluctuate due to environmental variablessuch as sunlight, ventilation, activity, and the “reservoir effect.” Forexample, DIPA-1-8 and 2-8 are exceptionally long-acting on the skin.

The effects of test compounds on periorbital skin, malar (zygomatic)skin, and forehead skin were determined.

Compounds were tested on periorbital skin. Test compounds were appliedto the closed eyelids using cotton gauze (0.4 g, rectangular, 50 mm×60mm; from CS-being, Daisan Cotton, Japan). The test compounds were usedat a concentration of 1 mg/mL in distilled water. The duration of thesensory effect was measured with a stopwatch. The degree of “dynamiccool” was graded from 0 to +++, with intermediate steps of + and ++. Ananti-fatigue effect was present only if there was sufficient “dynamiccool.”

The results are summarized in the following table.

Carbon Sensory Anti- Duration Sting on Code R₃ atoms Quality fatigue(hr) Ocular Surface 1-5 5 11 dynamic + 0.5 No DIPA- 6 12 dynamic ++ 3.8Yes 1-6 DIPA- 7 13 dynamic +++ 4.2 No 1-7 DIPA- 8 14 cool ++ 2.1 No 1-8DIPA- 9 15 cool 0 3.0 No 1-9 2-4 4 12 cool 0 0.1 No 2-5 5 13 cool + 2.1No 2-6 6 14 cool ++ 6.2 Yes 2-7 7 15 cool + 1.2 Yes 2-8 8 16 cool + 1.3No

Compounds were tested on zygomatic and forehead skin. Test compoundswere applied to the skin of the forehead and zygomatic using cottongauze (0.4 g, rectangular, 50 mm×60 mm; from CS-being, Daisan Cotton,Japan). The test compounds were used at a concentration of 20 mg/mL indistilled water. The onset and duration of the sensory effect wasmeasured with a stopwatch. The degree of “dynamic cool” was graded from0 to +++, with intermediate steps of + and ++. An anti-fatigue effectwas present only if there was sufficient “dynamic cool.” The results aresummarized in the following table.

Carbon Onset Sensory Anti- Duration Reservoir Code R₃ atoms (min)Quality Fatigue (hr) Effect DIPA-1-5 5 11 ~1 dynamic 0 0.5 No DIPA-1-6 612 ~1 dynamic ++ 1.3 Yes DIPA-1-7 7 13 ~1 dynamic- +++ 3.2 Yes icyDIPA-1-8 8 14 ~1 cold-icy ++ 4.0 Yes DIPA-1-9 9 15 ~2 cool 0 2.0 No 2-44 12 ~1 cool 0 0.3 No 2-5 5 13 ~1 cool 0 1.1 Yes 2-6 6 14 ~2 cold + 1.5Yes 2-7 7 15 ~2 cold + 2.4 Yes 2-8 8 16 5 cold 0 5.6 Yes

Each of 3-1 and 3-2 was tested and found to be inactive on periorbital,and zygomatic/forehead skin.

Notably, DIPA-1-7 selectively produced the unusual sensation of “dynamiccool” and also had anti-fatigue effects. From the data shown above, itcan be seen that, among these compounds, DIPA-1-7 evoked “dynamic cool”on both periorbital and zygomatic/forehead surface. Another compoundwith similar properties was DIPA-1-8, but this compound is was morecold/icy cold, although it had the desirable property of a longerduration of action on the zygomatic/forehead surface. The long durationof action of DIPA-1-7 and DIPA-1-8 on the skin adds value as ananti-fatigue agent, especially for the fatigue of chronic illness. Asshown in the case studies described below, a single application ofDIPA-1-7 is sufficient to counteract fatigue and heat stress for atleast three to four hours.

A special value of DIPA-1-9 is the comfortable cooling it provides andits long duration of action after periorbital application, and theabsence of any stinging. Thus, it has a special therapeutic niche forthe relief of ocular discomfort.

A study of structure-activity relationships did not reveal anyattributes of DIPA-1-7 that would have predicted its unique properties.For example, dynamic cool is seen with 2-5 on the oropharyngeal surface,but 2-5 does not elicit this sensation when applied the skin with awipe.

The sensory properties of the anti-fatigue effects of DIPA compounds andtheir duration of action could not have been predicted based on standardcorrelations of lipophilic and hydrophilic parameters. For the durationof action on the zygomatic/forehead skin, increasing the number ofcarbons on R3 increased the duration of cooling, as might be predictedon the basis of lipophilicity, but the periorbital effects indicatehydrophilicity is also important for anti-fatigue actions. In thesection on “Receptor Mechanisms”, the importance of a partial charge onthe phosphinoyl oxygen for hydrogen bonding and an “on-off” or “rapidassociation-dissociation” for activating dynamic cool is discussed. Theresults here for the selective attributes of DIPA-1-7 and DIPA-1-8 areunexpected, surprising, and has practical applications forcounter-acting fatigue and anti-nociception.

Study 4 Agonist Activity of Compounds on TRPM8

The in vitro effects of test compounds were evaluated on cloned hTRPM8channel (encoded by the human TRPM8 gene, expressed in CHO cells) usinga Fluo-8 calcium kit and a Fluorescence Imaging Plate Reader(FLIPRTETRA™) instrument. The assays were conducted by ChanTestCorporation (14656 Neo Parkway, Cleveland, Ohio 44128, USA).

Test compounds and positive control solutions were prepared by dilutingstock solutions in a HEPES-buffered physiological saline (HBPS)solution. The test compound and control formulations were loaded inpolypropylene or glass-lined 384-well plates, and placed into the FLIPRinstrument (Molecular Devices Corporation, Union City, Calif., USA). Thetest compounds were evaluated at 4 or 8 concentrations with n=4replicates per determination. The positive control reference compoundwas L-menthol, a known TRPM8 agonist. The test cells were ChineseHamster Ovary (CHO) cells stably transfected with human TRPM8 cDNAs.

For FLIPRTETRA™ assay, cells were plated in 384-well black wall, flatclear-bottom microtiter plates (Type: BD Biocoat Poly-D-Lysine MultiwellCell Culture Plate) at approximately 30,000 cells per well. Cells wereincubated at 37° C. overnight to reach a near confluent monolayerappropriate for use in a fluorescence assay. The test procedure was toremove the growth media and to add 40 μL of HBPS containing Fluo-8 for30 minutes at 37° C. 10 μL of test compound, vehicle, or controlsolutions in HBPS were added to each well and read for 4 minutes.

Concentration-response data were analyzed via the FLIPR Control softwarethat is supplied with the FLIPR System (MDS-AT) and fitted to a Hillequation of the following form:

${RESPONSE} = {{Base} + \frac{{Max} - {Base}}{1 + \left( \frac{xhalf}{x} \right)^{rate}}}$

where: “Base” is the response at low concentrations of test compound;“Max” is the maximum response at high concentrations; “xhalf” is theEC₅₀, the concentration of test compound producing half-maximalactivation; and “rate” is the Hill coefficient. Nonlinear least squaresfits were made assuming a simple one-to-one binding model. The 95%Confidence Interval was obtained using the GraphPad Prism 6 software.The results are summarized in the following table.

Code EC50 μM 95% Confidence Interval Relative Potency Menthol 3.8 2.5 to5.6 1.0 1-5 5.6 4.4 to 7.2 0.7 DIPA-1-6 2.4 1.5 to 4.0 1.6 DIPA-1-7 0.70.5 to 1.0 5.4 DIPA-1-8 0.7 0.5 to 1.0 5.4 DIPA-1-9 0.9 0.4 to 2.5 4.02-4 14.5   7 to 29 0.3 2-5 1.7 1.0 to 2.9 2.2 2-6 0.8 0.5 to 1.3 4.7 2-71.1 0.6 to 2.3 3.4 2-8 1.3 0.7 to 2.3 2.9 3-1 24    8 to 76 0.2 3-2 4.2 1.6 to 10.8 0.9

All of the compounds were found to have full efficacy on the receptor:that is, there is up to 100% activation, and the dose levels tested fitinto a sigmoidal dose-response relationship.

The results for the “di-isopropyl” compounds are illustrated in FIG. 2.

FIG. 2 is a graph of response (Relative Fluorescence Units; % ofmaximum) as a function of the logarithm of the concentration of the testcompound (denoted agonist), expressed in μM, for each of 1-5 (circles),DIPA-1-6 (squares), DIPA-1-7 (inverted triangle), DIPA-1-8 (diamonds),or DIPA-1-9 (up-right triangle).

DIPA-1-7 and DIPA-1-8 are significantly more potent than 1-5 andDIPA-1-6. The 95% confidence intervals of DIPA-1-7 and DIPA-1-8 aresimilar with overlapping 95% confidence intervals. DIPA-1-7 is moreeffective at producing the sensation of “dynamic cool” on the skin andon the ocular surface. Also, the potencies of DIPA-1-7 and DIPA-1-8 aresignificantly greater than the potencies of 1-5 and DIPA-1-6.

Of the 12 compounds tested, all showed full efficacy on the TRPM8receptor, i.e., at higher tested concentrations there was ^(˜)100%stimulation of calcium entry, and the data fitted a sigmoidaldose-response curve. The EC50 of the more potent compounds (DIPA-1-6,DIPA-1-7, DIPA-1-8, DIPA-1-9, 2-5, 2-6, 2-7, 2-8) fell within a narrowrange with overlapping 95% Confidence Intervals. There were nodistinguishing features in the EC50 data which enabled prediction ofwhich compounds have “dynamic cool” properties. The structuralmodifications of 3-1 and 3-2 resulted in a significant loss ofbioactivity.

Study 5 Studies on Isolated Vagus Nerve: Direct Anti-NociceptiveActivity

To determine if DIPA-1-7 acted directly on sensory nerves, it was testedin an isolated nerve model developed at the Imperial College, London,U.K. (see, e.g., Birrell et al., 2009; Patel et al., 2003). In this invitro assay, segments of the mouse vagus nerve are placed on a platformand the electrical activity is recorded after topical application ofcapsaicin. Capsaicin is a known irritant that elicits pain when it isapplied to the skin and it will depolarize the isolated vagus. Theability of substances to inhibit this capsaicin-induced depolarizationis measured.

Briefly, segments of vagus nerve, caudal to the nodose ganglion, wereremoved from mice with fine forceps and segments placed in oxygenatedKrebs solution and bubbled with 95% O2/5% CO2. The desheathed nervetrunk was mounted in a ‘grease-gap’ recording chamber and constantlysuperfused with Krebs solution with a flow rate of approximately 2mL/min, and the electrical activity of the nerve monitored withelectrodes. The temperature of the perforate was kept constant at 37° C.by a water bath. Nerve depolarizations were induced by superfusion ofthe nerve with capsaicin (1 μM). After two reproducible depolarizationresponses to capsaicin, DIPA-1-7 was applied at 1 mg/mL (4 μM) for 10minutes in the perfusate followed by capsaicin. The nerves were thenwashed with Krebs until the responses had returned to baseline andchallenged again with capsaicin. The results and tracings obtained innormal and TRPM8 knockout mouse are shown in FIG. 3.

FIG. 3 shows chart traces that illustrate, in the first trace (“WildType”), the inhibition of capsaicin-induced depolarization of theisolated mouse vagus by DIPA-1-7, superfused at a concentration of 1mg/mL, and, in the second trace (“TRPM8 KO”), the significant absence ofinhibition in the isolated TRPM8 KO (knockout) mouse vagus by DIPA-1-7,superfused at a concentration of 1 mg/mL.

In the tracings shown in the figure, the first two peaks show thedepolarization response of the mouse vagus to capsaicin (“Caps”). AfterDIPA-1-7 is applied (1 mg/mL), the response is suppressed in the normalmouse vagus (“Wild Type”), but not in the TRPM8 knock-out (“TRPM8 KO”)mouse vagus.

The percent inhibition of capsaicin-induced depolarization of theisolated normal mouse vagus caused by DIPA-1-7 was about 75%; thepercent inhibition of capsaicin-induced depolarization of the isolatedTRPM8 knock-out mouse vagus caused by DIPA-1-7 was about 20%.

This experiment clearly demonstrates a direct pharmacological action ofthe DIPA-1-7 on the sensory nerve, which is a surprising and unexpectedresult. Furthermore, the diminished response in the TRPM8 KO mouseindicated that the receptor target was TRPM8. These results providestrong evidence that DIPA-1-7 can be used as an anti-nociceptive agentand the target receptor is TRPM8.

Capsaicin is a TRPV1 agonist and the search for an effective TRPV1antagonist has been the super-intense quest of many pharmaceuticalcompanies for the past ten or more year. Here, it is shown that DIPA-1-7is an effective “physiological” antagonist of TRPV1 at lowconcentrations. DIPA-1-7, by itself, did not evoke depolarization,indicating that it is free of agonist activity at this “pain” receptor.These results strongly indicate the usefulness of DIPA-1-7 as ananti-nociceptive agent.

Study 6 Bioactivity in Laboratory Animals

Fur-coated and feathered animals—when wet and cold—shake, like a wet dog(see, e.g., Dickerson et al., 2012; Ortega-Jimenez et al., 2012; Wei,1981). These shakes are rapid alternating contractions of the supinationand pronation muscles about the spinal axis, and can be readily observedand counted. “Wet-dog shaking” has been studied in detail in animals andthis behaviour is interpreted to have survival value because shaking, byremoving the water off its skin, reduces the need to expend evaporativeenergy to remove wetness. The triggering sensation for shaking is thushaving water trapped in between hair follicles or feathers. Humans havelittle hair on skin and do not shake. The likely equivalent behaviour toshaking in humans is shivering, a condition caused by generalizedsensations of coolness/cold and wetness.

Drug-induced shaking in animals has been reviewed (see, e.g., Wei,1981). Under the right conditions, drug-induced shaking can be observedin the pentobarbital-anesthetized rat, and enhanced by hypothermia andcold.

Test compounds were evaluated for “wet-dog shaking” as a model ofdynamic cooling. Using a standardized procedure, test compounds werecompared in their ability to stimulate the shaking response. 20 mg/kg ofeach test compound was administered by oral gavage topentobarbital-anesthetized male albino rats. Shaking was counted over a40 minute period at 10-minute intervals. The data are summarised in thefollowing table.

No. of Topical Sensory No of carbon Quality on shakes in Code R₁ R₂ R₃atoms zygomatic skin 40 minutes DIPA-1-5 iPr iPr pentyl 11 dynamic cool86 ± 7 DIPA-1-6 iPr iPr hexyl 12 dynamic cool 56 ± 5 DIPA-1-7 iPr iPrheptyl 13 dynamic cool 36 ± 4 DIPA-1-8 iPr iPr octyl 14 cool 0 DIPA-1-9iPr iPr nonyl 15 mild cool 0 2-4 sBu sBu butyl 12 cool 0 2-5 sBu sBupentyl 13 cool  4 ± 1 2-6 sBu sBu hexyl 14 cool 0 2-7 sBu sBu heptyl 15cool 0 2-8 sBu sBu octyl 16 cool 0

Three of the four “di-isoproypyl” compounds caused vigorous shaking. The“di-secbutyl” compounds were relatively inactive, except 2-5 whichelicited an average of 4 shakes in the 40 minute observation period. Bycontrast, 1-5, DIPA-1-6, and DIPA-1-7 produced an average shakingfrequency of 86, 56, and 36 shakes, respectively. The strong activity of1-5 was unusual. Applied to the skin, 1-5 has a refreshing “dynamiccool”, but the duration of action of only about 30 minutes wassignificantly less than that for DIPA-1-6 and DIPA-1-7. The shorterduration of action of 1-5 limits its practical utility. It is possiblethat its smaller molecular size facilitates absorption and allowsgreater access to target receptors, and therefore more shaking.

These results provide the strongest objective laboratory evidence thatsome of the compounds selectively produce vigorous “dynamic cool” andsome do not. The total number of carbons in the compound, or the numberof carbons in the largest alkyl group, did not appear to be a criticaldeterminant of activity.

The relationship of the shake response to temperature sensation wasfurther studied in pentobarbital-anesthetized rats. After injection ofthe anaesthetic, rectal temperature drops, and reaches approximately 35°C. about 10 minutes after the onset of anaesthesia. This can be reversedby placing the animal on a heated surface and the body temperaturemaintained at 38° C.

20 mg/kg of DIPA-1-7 was administered by oral gavage topentobarbital-anesthetized male albino rats. Shaking was counted over a40 minute period at 5- or 10-minute intervals. In the non-heatedanimals, after 40 minutes, DIPA-1-7 elicits 36±5 shakes (N=6). In theheated animals, the shaking frequency is significantly reduced to 5±2shakes (N=6).

This study shows that shaking induced by DIPA-1-7 is inhibited by heat.The number of shakes evoked by DIPA-1-7 was reduced by ⅔ when theanesthetized rat was placed on a warm surface and body temperaturemaintained at 38° C. Thus, the frequency of shaking is counter-acted byheat, indicating its link to cold sensations and shivering.

Study 7 Effects on Topical Sites on the Cranium

DIPA-1-7, the most potent compound for dynamic cooling, was tested atother topical sites on the cranium. A 20 mg/mL solution was applied,using a cotton wipe, onto the skin above the buccal cheek, theparotid-masseteric cheek, temple, and the skin above the periauricularregion, and the posterior mandible using the appropriate craniometricpoints (pterion, coronion, condylion, and gonion, respectively) aslandmarks. Surprisingly, at all of these sites, other than the buccalcheek, little cooling, if any, was observed. Mild cooling was observedon the buccal cheek for approximately 30 minutes, but this effect mayhave been due to the spread of the solution onto the receptive field ofthe infraorbital nerve. Thus, the action on orbit and zygomatic/foreheadskin is selective and identifies the important delivery targets on theskin of the head.

The head is known to be a site where cooling helps relieve heatdiscomfort. In a study described in Nakamura et al., 2012, eleven malesubjects were exposed to mild heat. Subjects, clothed in only shortpants, entered a climatic chamber maintained at 32.5±0.5° C. with arelative humidity of 50%. About 1.5 hours after entry into the chamber,a local cooling protocol was initiated with water-perfused stimulatorsplaced on the head, chest, abdomen, or thigh. Cooling of the face andthigh was felt by the subjects to be more effective than cooling of thechest and abdomen in reducing the heat discomfort.

In a study described in Essick et al., the thresholds for detection ofcooling and cold pain on various sites of the face, ventral forearm, andscalp was determined for 34 young adults. The most sensitive sites wereon the vermilion which could detect a temperature change of about 0.5°C., followed by areas around the mouth (upper and lower hairy lip, mouthcorner) and lateral chin. The mid-cheek and periauricular skin were lesssensitive (able to detect a temperature change of about 2° C.), and theforearm and scalp were least sensitive (able to detect a temperaturechange of about 3° C.). The sensitivities of the orbital, zygomatic andforehead skin were not tested.

Use of DIPA-1-7 on the orbital and zygomatic/forehead skin, for example,in an office environment or in heat stress, may be inconvenient if thesubjects uses cosmetic make-up at these sites. Surprisingly, it wasfound that DIPA-1-7, at 20 mg/mL, can produce a dynamic cooling effectwhen applied on the scalp, especially near the hairline. This effect issufficient to counter fatigue caused by heat. Likewise, rubbing DIPA-1-7on the skin in the centre of the chest, above the sternum, cancounteract the discomforts of heat. At these application sites,cosmetics are not affected, yet an invigorating coolness, thatcounteracts the debilitating effect of heat, is achieved.

The ability of DIPA-1-7 to cause cooling of the scalp and hairline isalso important for treating itch at these sites in conditions such aspsoriasis, dandruff, and seborrheic dermatitis.

Case Studies

Case studies are described below which demonstrate the use of DIPA-1-7:(a) to enhance cognition, decrease mental lassitude and fatigue, and toenergize performance; (b) to counteract tiredness and fatigue fromchronic illness; (c) to counteract the fatigue and/or discomfort fromheat stress; (d) to counteract skin itch and pain, and (e) to reduce theseverity of “night sweats”.

In these studies, subjects were given dosages units containing 1.5 to1.75 mL of DIPA-1-7 stored in 2.0 mL microcentrifuge tubes (NovaBiostorage Plus, Canonsburg, Pa. 15317) and cotton gauze (0.4 g,rectangular, 50 mm×60 mml; from CS-being, Daisan Cotton, Japan). TheDIPA-1-7 was provided as a solution in distilled water or 2% ethanol-98%distilled water, at a DIPA-1-7 concentration of 1 mg/mL or 5 mg/mL. Thesubjects were given instructions on how to place the solution on thegauze and how to wipe the wet gauze over the skin surfaces with the eyesclosed: 5 mg/mL for the orbital and zygomatic/forehead skin, away fromthe palpebral sulcus, and 1 mg/mL if the primary site was theperiorbital skin. Approximately 0.35 mL and 0.15 mL are delivered bythese methods of application, respectively.

For some test compounds (e.g., 2-6 and 2-7), residues that remain onorbital skin can enter the ocular surface and cause stinging anddiscomfort when a subject sweats or takes a shower. This problem wasminimal with DIPA-1-7 and DIPA-1-8. Subjects were instructed to rinsewith water or a wet towel any surfaces that become irritable; however,irritation and discomfort was rarely seen with DIPA-1-7 or DIPA-1-8 atthese concentrations.

Case Study 1

A 65-year old male is an avid snooker player and likes to frequent thesnooker parlours of London and Hong Kong. He plays for small wagers withhis friends, but with advancing age his game has deteriorated and he canonly play about eight frames in one day. He uses ice-cold towels on hisface and prescription glasses to help him during games, but feels thatit is the lack of concentration and the planning of sequences of shotsthat hinders his game and prevents him from completing “breaks” (acontinuous accumulation of points in a “run”). He volunteered to trywipes containing DIPA-1-7. There was a remarkable transformation in hisgame. He moved faster from shot to shot and the planning and executionwas crisp. The number of frames per session increased as well as hisfrequency of play. He had his longest career break of 80 points and wasecstatic. He continues to use the wipes as an aid to his snooker game.He also noted that enhancement of his cognitive facilities could berenewed and invigorated by applying the ice cold towel to his face (anexample of the “reservoir effect”). He noted, however, it was importantto avoid excessive entry of the DIPA-1-7 onto his ocular surface becausethat sometimes caused irritation, especially if the use was toofrequent. With practice, he noted that cognitive enhancement of his gamecould be regulated and controlled by optimizing the delivery procedures.

A 70-year old retired architect likes to play penny poker once or twicea week with his buddies. He volunteered to try wipes containing 5 mg/mLof DIPA-1-7 to see if it would improve his poker skills. He did this atfirst without telling his friends. He immediately noticed afterapplication of the wipe that he was more awake than the other players.He could remember the cards that were discarded, could calculate andremember the odds of various hands (e.g., likelihood of drawingsuccessfully to a four-card two-way straight or a four-card flush), butmost importantly, he could also sense if his opponent had a strong orweak hand, and if they were bluffing. He felt energized, moreadventurous, and willing to take risks by bluffing himself. He madedecisions quickly and with more confidence. He felt that his game wasmore insightful and improved. He felt guilty about having an unfairadvantage over his friends and encouraged several of the other playersto try the wipes. All noticed the invigorating dynamic cool sensationsbut they were less sure if their poker skills were improved.

A 68-year old pharmacologist spends his time in research and in thedesign and management of clinical trials. He owns his consulting firmwith eight employees, and spends at least 8 to 12 hours per day in frontof a computer monitor. He has in his working space an espresso machine,and boxes of cigarettes and cigars. He uses coffee and tobacco tosharpen his thinking. He agreed to apply the wipes containing DIPA-1-7at 1 mg/mL (periorbital only) and 5 mg/mL (periorbital andzygomatic/forehead) and noted that his tiredness went away for at least6 to 8 hours and that he was able to concentrate and think more clearly.He said the wipes were superior to both coffee and tobacco in improvinghis concentration. He now also uses the wipes for work and beforebusiness and scientific meetings to enhance his social performance andmental acuity, and to reduce fatigue.

A 72-year old retired policeman decided to return to work as a securityguard because he needed the funds to support his grand-daughter'scollege costs. He worked from noon to 8:30 pm and complained ofweariness and fatigue which affected his activities. He said he was sotired that he could not stay awake for televised football games, eventhough he was an avid fan. He volunteered to try the wipes containingDIPA-1-7 and said they definitely made him more vigilant, especiallywhen driving home from work. He said that turning on the car'sair-conditioning so that the cool air vent was aimed at his face,together with menthol mints and the wipes, kept him alert, and that heno was no longer a threat on the roads. He had an 18.5 inch (47 cm)neckline and snored heavily at night, but polysomnography did not revealsleep apnea episodes. He felt that by using the wipes on his orbit somecoolness drained down onto his nasal membranes (via the nasolachrymalduct), and that this cooling sensation in his nose allowed him tobreathe more freely and to sleep better at night. Currently, he isexercising more and trying to reduce food intake, in order to controlhis fatigue.

Several individuals also tried the wipes containing DIPA-1-6, DIPA-1-8,2-6 and 2-7, and also found these compounds to be effective forenhancing performance and thinking, but the effects were consideredsomewhat less dramatic, or with some residual sting. Of these analogs,DIPA-1-8 was judged to be the best alternative to DIPA-1-7 for cognitiveenhancement. In is possible, with the appropriate formulation, all ofthese analogs might be used as alternatives. In summary, the surprisingobservation made here was that use of these compounds, and in particularDIPA-1-7, can enhance skills requiring hand-eye coordination (e.g., insnooker) and concentration (e.g., in games of chance such as poker).

Case Study 2

A 48-year old female account executive was a busy professional at alarge financial institution. Her husband was a successful architect. Shehad two teenage children and she was constantly short of time to do herchores. At the end of the day, she was frequently physically andmentally exhausted and would fall asleep early after evening meals. Dueto recent marital difficulties, she felt tired and weary most of thetime, and her domestic and professional demeanour, in dress andetiquette, began to deteriorate. She did not suffer from any chronicphysical illness, but she was rated as having “moderate fatigue” on theBrief Fatigue Inventory (BFI) after several interviews and considered“depressed” by her physician. She volunteered to use the wipescontaining DIPA-1-7 and was instructed not to use more than one per day.After two days of use, she reported that the wipes improved her mood andinterest in external events. She was more energetic and positive. Shecompleted her assignments at work promptly and had better stamina, andshe was more combative and assertive. The people closest to her,children and work colleagues, also remarked on her improved change inattitude and personality. She continues to use the wipes on an as-neededbasis.

A 69-year old male suffered from Parkinson's disease of 12 yearsduration. He is on expert medical care and has taken a variety of drugsto help manage his disease over time. In the past several years, theprimary drugs (e.g., Sinemet®) became less effective and he was lessmobile and more housebound. In November 2009, he was implanted withelectrodes for deep brain stimulation treatment and this procedureincreased his mobility. Recently, however, in spite of carefuladjustment of his brain stimulation parameters, his Parkinsonism hasgradually returned, and he complained of constant fatigue and depressedmood. His BFI scores were in the “moderate to severe” range of fatiguelevels. He volunteered to try the wipes containing DIPA-1-7 (1 mg/mL and5 mg/mL) and was told to limit his use to one per day. The first thingthat the subject noticed, after using the wipes, was that he was able tostay awake and alert in order to watch his two favourite TV shows“House” and “Hawaii Five-0” on Monday nights (from 9 to 11 pm). He saidnormally he would have to make an extra effort to follow the dialogueand plot of “House” but would fall asleep before Hawaii Five-0 “gotgoing”. His general activity and mood improved and he was more willingto take his dog for a walk. He went to the golf range more often to dochipping and putting, but said he was still unable to turn to swinglonger clubs off the mat. His friends noticed he was in a better moodand participated more in social events. He attributes his reducedtiredness to the wipes and looks forward to its use every morning. Hesaid his appetite had improved, he longer felt depressed, and he wantedto be more active.

A 62-year old was diagnosed with hepatitis C virus (HCV) infection 10years ago and was treated with PEG-interferon and ribavarin but did notrespond because of his genetic makeup. He retired early from hisprofessional career and was relatively symptom-free except for mildfatigue which required a mandatory afternoon nap of at least two hours.However, six months ago, a 3 cm diameter hepatoma was detected bymagnetic resonance imaging on the margin of his lower right liver lobe.He was first treated by trans-arterial chemical embolization withdoxorubicin-eluting beads (TACE) and then shortly afterwards withradiofrequency ablation when it was noted that his α-fetoprotein levelswere elevated, suggesting that hepatoma cells may still be present afterTACE. These procedures resulted in moderate to severe fatigue, asevaluated by the BFI, which remained persistent even two months afterthe last treatment procedure. His initial complaint of severe pain aftersurgery was managed by the narcotic analgesic Vicodin®, but now his maincomplaint is of disturbed sleep, daytime fatigue, inability toconcentrate, and memory loss. He was prescribed the hypnotic Lunesta®,but this did not help his disturbed sleep, so he is now prescribedZolpidem®, despite the increased risks of liver damage from this drug.He volunteered to try the wipes containing DIPA-1-7 (1 mg/mL and 5mg/mL) because he was an avid reader, belonged to a book club, andwanted to keep his mind active when his mobility was physically limitedby fatigue.

After using the wipes, he commented that he was more alert and he wasbetter able to concentrate when reading. He noted that applying thewipes to a wider surface, especially on the skin of the cheekbones andorbit, enhanced the desired sensory effect. (The delivery of the sensoryagent to the neuronal receptive field is enlarged.) He noted that he hadfinished reading Kurt Vonnegut's biography but was discouraged fromtackling the biography of Steve Jobs by Walter Issacson because of itslength (more than 600 pages). After using the wipes, he finished readingthe Jobs biography in three days, and was able to remember and discussthe finer details of the book with his friends. He was especiallyintrigued by how Jobs was treated for and responded to his cancer. Hesaid his pain from surgery was not improved by use of the medicatedwipes, and he still had aches in his joints, but his mood and hisability to carry out daily activities were improved. He noted that theexceptionally long duration of action of the active ingredient in thewipes may be of use in treatment of other chronic illnesses suchnarcolepsy, neurotic and major depressions, and as an adjunct inmanaging Alzheimer's disease. He continues to use the wipes on anas-needed basis.

These studies illustrate the potential benefits of the medicated wipes,especially those containing DIPA-1-7, for countering the tiredness andfatigue of chronic illness.

Case Study 3

In another series of studies, a towelette was used for delivery insteadof a cotton wipe. The towelette consisted of a plastic wrap (weight 1.1g), a 23 cm×26 cm towel of non-woven lace (weight 3.4 to 3.5 g) and aliquid composition (14 to 15 mL) which was automatically added to andsealed off in the wrapper. Automated machinery for producing towelettesare well-known to the art. Here, the towelettes were produced by KankFactor, LLC, San Francisco (721 Commercial Street, San Francisco Calif.94108, www.3LWipes.com). Distilled water (as placebo controls) orDIPA-1-7 dissolved in distilled water (at a concentration of 1 to 5mg/mL) was incorporated into the towelette. The volume perself-application depended on the application site, but was about 0.3 mLto 0.5 mL for the face and brow, but could be higher if wiping of thetorso was also included.

The towelettes were stored in a refrigerator but then stored at roomtemperature for at least 1 hour before use. Effective sterilization ofthe towelette could be obtained by placement in a microwave oven for 1min (see, e.g., Tanaka et al., 1998). Subjects were instructed to holdthe towelette with both hands, and bring the towelette against the face,like how one would use a small wet face towel, and to keep the eyesclosed. The skin of the face is moistened and medicated by thisprocedure. Once the subject has learned what to expect, the subject canadjust the dosage (e.g., by dabbing), as needed, to achieve the desiredanti-fatigue/anti-heat effects. After one or two trials, individualsquickly learn how to apply the sensory agent without any risks ofdiscomfort.

During an “Indian Summer” heat wave in the San Francisco Bay Area, theoutside temperature was 30 to 33° C. with a cloudless sky and an intensebright sun. The towelette, described above, was used as a substrate todeliver DIPA-1-7 to the skin of the chest and armpits of severalindividuals who complained vigorously about heat stress and discomfort.Comfortable cooling was noted for more than 3.5 hours with decreasedsweating. These Individuals were able to work normally in the heat in anoffice environment without need for additional cooling.

A 70-year old from Northern California went on a 7-day golf vacation toLas Vegas in September. He played at least one round of golf each dayand sometimes two. He did not wear a hat or use sunscreen. On the thirdday of vacation, the subject showed the classic signs and symptoms ofsunburn: redness and flushing of the facial skin, a sense of persistentwarmth, pain, and tenderness of the face, a mild degree of swellingaround the eyes, and a throbbing headache. He volunteered to try a creamcontaining 1% wt/vol DIPA-1-8 and wiped about 0.5 mL of the cream overhis cheeks and cheekbone. Surprisingly, he noted an immediate relief ofskin discomfort which lasted for at least four hours. His headache wasgone, and he said his face felt “comfortable and normal”. He used thecream on an “as needed” basis and also took measures to reduce hisexposure to direct sunlight by wearing a wide-brimmed hat and applyingcopious amounts of sunscreen products.

A second-year medical student in the American South was preparing forher Boards in the summer. During hot weather, her electricity costsincreased three-fold, so that she and her roommates could not afford toturn on the air-conditioning throughout the evening hours. She said thatshe could cope with the heat by using a wet towel around her neck, butthe main adverse effect of heat was disturbance of mental concentrationfor studying and the difficulty in getting comfortable sleep. She agreedto try the towellette containing DIPA-1-7 and found that it gave herprolonged and refreshing cooling sensations of her face and body. Sheremarked that her skin felt fresh and cool and she was better able toconcentrate of her studies and to retain information. She also notedthat her boyfriend said that she had a fresh and energetic look aboutthe eyes, like Julia Roberts in her younger days, and that this lookmade her more attractive. She said that DIPA-1-7 may have value as acosmetic agent to enhance beauty, as well as an aid to enhanceconcentration and study in an academic situation. She also noted thatDIPA-1-7 might be useful in the same way that icy collars put around theneck significantly improve athletic performance.

Case Study 4

Two scientists working in the laboratory had allergic dermatitis of thehand in response to detergents and soaps. The hands were inflamed andextremely itchy. Applications of DIPA-1-7, 20 mg/mL, with acotton-tipped applicator immediately stopped the itch and this effectlasted for at least 2 hours, and the suppression could be renewed byrepeated application. One scientist, a world-renowned dermatologist withmany publications on itch, noted that the DIPA-1-7 produced an“icy-cool” feeling on the inflamed skin and he had never encounteredsuch a compound that was so effective in stopping itch so quickly.

A pharmacologist liked to work in the garden, but the thorns frombougainvillea stems and rose bushes, and the hair from azalea leaves,irritated his skin and caused intense itch. He noted that the sensorydiscomfort on the skin could be instantly stopped by DIPA-1-6 orDIPA-1-7, applied either as a 20 mg/mL aqueous solution, or as a cream(mixed with Eucerin Moisturizing Cream). These effects could also beobtained with DIPA-1-8. He also noted that the irritation and itchcaused by insect bites could be immediately stopped by these agents.

A 40-year old suffered from penile lichen sclerosus. This is aninflammatory dermatosis of the glans penis and foreskin and, in thisparticular case, was associated with intense pruritus and dysesthesias(burning sensations). The patient, under the supervision and care of hisdermatologist, volunteered to try DIPA-1-8 on his lesion and he wassupplied with various concentrations of DIPA-1-8 dissolved in distilledwater. After self-experiment, he concluded that concentrations of 1 to1.5 mg/mL of DIPA-1-8 produced significant relief, but a concentrationof 2 mg/mL of DIPA-1-8 was too cold and uncomfortable. The solutionswere applied with cotton-tipped applicators or gauze wipes. Theadvantage of using DIPA formulations for genital skin is watersolubility. This minimizes the need for excipients and the likelihood offurther irritation. The subject suggested that an aerosolized spray mayalso be a convenient method of drug delivery.

These studies illustrate the anti-nociceptive properties of DIPA-1-7 andDIPA-1-8, especially on itching. DIPA-1-8 had a longer duration ofaction than DIPA-1-7, and may be the preferred agent for dermatologicalapplications.

Case Study 5

A 66-year old woman had occasional bouts of hot flushes/night sweats ofabout 1 episode every two weeks. She was on hormone replacement therapy(HRT) (estradiol 1 mg and medroxyprogesterone 2.5 g, once per day), butdecided to stop HRT after two of her friends had breast cancer and onehad uterine cancer. Her episodes of night sweats increased to about onceevery other day, and she and her husband were frustrated because it wasnecessary to change the bed sheets frequently. She agreed to try alotion containing 1% of DIPA-1-6. This lotion was applied on the skin atthe base of her neck and on the centre of her chest before going tosleep at night. If she woke up at night, the application was repeated.She said the lotion felt cool, but was not uncomfortable. No episodes ofnight sweats were observed for three weeks. Further discussion with herphysician convinced her to return to HRT and she has not experiencednight sweats for at least the past 9 months.

Case Study 6

Three subjects decided to systemically compare DIPA-1-6, DIPA-1-7,DIPA-1-8, and DIPA-1-9 for their sensory effects on the ocular surface.Each compound was prepared at 1 mg/mL in distilled water. A cottontipped applicator of a specific size (Puritan 803-PCL) consisting of a55 to 75 mg ball of cotton wound around the tip of a three inchpolystyrene rod was dipped into the solution. The tip was then applied,with the eyelids closed, to the lower aspect of the upper eyelid, ontothe eyelashes, with two lateral to medial wiping motions. The subjectswere then instructed to blink. By blinking, the solution is then evenlydistributed over the pre-corneal film. This “swab” delivery methodoff-loaded a total of ^(˜)35 μL of liquid onto the surface of both eyes.DIPA-1-6 caused significant stinging and discomfort and was thereforenot further studied. DIPA-1-7 and DIPA-1-8 produced strong andrefreshing cooling, which counter-acted eye irritation, and increasedcognitive functions. For example, subjects felt they could focus ondistant objects and enjoy the view. They felt mentally alert andrefreshed. But, with both DIPA-1-7 and DIPA-1-8, there was a smallresidue left on the eyelid; subsequently using a towel to wash the facecan cause eye irritation. Surprisingly, DIPA-1-9 did not produce any eyeirritation when wiped over the eyelid, nor did it leave a residue. Italso produced refreshing cooling, but not with the same intensity asDIPA-1-7 or DIPA-1-8. On the other hand, DIPA-1-9 has ideal propertiesfor the treatment of ocular discomfort, e.g., discomfort caused by eyestrain; eye fatigue; eye surgery; an airborne irritant or pollutant thatinteracts with the eye surface; extended wear of contact lenses;excessive exposure to the sun; conjunctivitis; or the dry eyes syndrome.

Case Study 7

A 2-year old female West Highland Terrier developed, during the summer,an itching condition which led to continued scratching of the ears andunderbelly. The veterinarian diagnosed the behavior as canine atopy andprescribed oral antihistamines. These drugs did not control theprogression of the itching and patches of raw skin, with hair loss,occurred at the base of the tail and on the hind limbs. A topicalanti-inflammatory steroid, triamcinolone, provided limited success andthe dog still looked miserable. Surprisingly, application of DIPA-1-7cream (1% wt/vol) to the inflamed skin sites immediately reducedscratching and the skin sites begin to heal. It was clear from the dog'sbehavior that the itching was reduced in severity. Further curtailmentof the dog's access to the outdoors and control of possible exposure tofleas and dust mites resulted in a successful control of the dog's skindisorder.

REFERENCES

A number of publications are cited herein in order to more fullydescribe and disclose the invention and the state of the art to whichthe invention pertains. Full citations for these publications areprovided below. Each of these publications is incorporated herein byreference in its entirety into the present disclosure, to the sameextent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

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1. A compound selected from the following compounds:


2. The compound of claim 1, wherein the compound is


3. The compound of claim 1, wherein the compound is


4. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier or diluent.
 5. The pharmaceuticalcomposition of claim 7, wherein the compound is contained in thecomposition at a concentration of 0.005-2.0% wt/vol.
 6. Thepharmaceutical composition of claim 7, which is a liquid composition andcomprises the compound at a concentration of 0.5-20 mg/mL.
 7. Thepharmaceutical composition of claim 7, wherein the compound is containedin the composition at a concentration of 1-5 mg/mL.
 8. Thepharmaceutical composition of claim 7, wherein the compound is containedin the composition at a concentration of 5-10 mg/mL.
 9. Thepharmaceutical composition of claim 7, wherein the compound is containedin the composition at a concentration of 10-20 mg/mL.